Proliposomal testosterone formulations

ABSTRACT

Novel testosterone formulations are disclosed where testosterone is incorporated into a phospholipid/cholesterol system to produce a proliposomal powder dispersion. The proliposomal powder dispersions of the invention may be formulated with pharmaceutically acceptable excipients to form pharmaceutical compositions. Enterically coated oral dosage forms are disclosed as are methods of treatment for testosterone replacement therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. patent application Ser. No.14/604,985, filed Jan. 26, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/149,227, now U.S. Pat. No. 8,957,053, filed Jan.7, 2014, which is a continuation of PCT International Application NumberPCT/US2013/040325, filed May 9, 2013, which claims priority to U.S.Provisional Application No. 61/644,996, filed May 9, 2012.

FIELD OF THE INVENTION

Disclosed herein are proliposomal pharmaceutical formulations for thedelivery of testosterone that increase testoterone's solubility andbioavailability.

BACKGROUND

Testosterone is a BCS class II drug. Unless otherwise indicated, theterm “testosterone” means testosterone that is chemically identical tothe testosterone produced by the human body, i.e, “native,” or“endogenous” testosterone. Testosterone may be isolated form naturalsources or made by commercial synthetic processes.

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, are proliposomal powderdispersions comprising (a) testosterone, (b) cholesterol, and (c) atleast one phospholipid. In some embodiments, (a) and (b) are present ina weight ratio (a):(b) ranging from 1:0.05 to 1:0.30 and (a), (b) and(c) are present in a weight ratio of (a):((b)+(c)) ranging from 1:1 and1:2.5. In some embodiments, (a) and (b) are present in a weight ratio(a):(b) ranging from about 1:about 0.05 to about 1:about 0.30 and (a),(b) and (c) are present in a weight ratio of (a):((b)+(c)) ranging fromabout 1:about 1 and about 1:about 2.5.

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising (a) a proliposomal powder dispersion describedherein, and (b) at least one pharmaceutically acceptable excipient.

Disclosed herein, in certain embodiments, are oral dosage formscomprising a pharmaceutical composition. In some embodiments, the oraldosage form is a tablet or capsule comprising (a) a proliposomal powderdispersion described herein and (b) at least one pharmaceuticallyacceptable excipient. In some embodiments, the tablet or capsule iscoated with a delayed release coating (e.g., an enteric coating). Insome embodiments, the tablet or capsule is coated with an entericcoating.

Disclosed herein, in certain embodiments, are oral testosterone dosageforms having fasting pharmacokinetic profiles that are characterized bya mean plasma concentrations of testosterone that range from about 350to about 950 ng/dL of testosterone at about five hours after ingestionof the dosage forms. The foregoing mean plasma concentration mayalternatively reported as ranging from about 4 to about 7 ng/dL/mg oftestosterone at five hours after ingestion. In some embodiments, thefasting pharmacokinetic profile is characterized by a mean plasmaconcentration of testosterone that ranges from 350 to 950 ng/dL oftestosterone at five hours after ingestion of the dosage form.

Disclosed herein, in certain embodiments, are methods of testosteronereplacement therapy comprising administering a proliposomal powderdispersion disclosed herein, a pharmaceutical composition describedherein, or a dosage form described herein to an individual in needthereof. Accordingly, disclosed herein are methods of treating anindividual in need of testosterone therapy comprising the step ofadministering to the individual a therapeutically effective amount of aproliposomal powder dispersion disclosed herein, a pharmaceuticalcomposition disclosed herein, or dosage form disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 exemplifies the in vitro testosterone dissolution profiles ofencapsulated uncoated PLF-C2 that: (a) had not been stored; (b) storedfor three months at room temperature; and (c) stored for three months at30° C. See Comparative Example 2.

FIG. 2 exemplifies the in vitro release profiles of testosterone fromuncoated capsules of PLF-C2 and PLF-C4 (which were prepared usingdifferent ratios of DPMG to the EtOH:water solvent solution) based ondissolution studies performed in PBS at pH 6.80. See Comparative Example2.

FIG. 3 exemplifies the mean plasma testosterone concentrations over timefollowing oral administration of a suspension of PLF-C1 and anunformulated control testosterone suspension to non-fasted rats. SeeComparative Example 3.

FIG. 4 exemplifies the mean plasma testosterone concentrations over timefollowing oral administrations of a suspension of PLF-C2 and asuspension of PLF-C3 to fasted rats. See Comparative Example 3.

FIG. 5 exemplifies the in vitro testosterone dissolution profiles of:(a) PLF-C5; (b) PLF-C5 with Avicel® PH101; (c) PLF-C5 with Avicel® PH101and Explotab® disintegrant; (d) hydrated PLF-C2; (e) PLF-C6; and (f)lyophilized PLF-C5. See Comparative Example 4 and Table 3.

FIG. 6 exemplifies solubility of testosterone after 72 hours in: (a) HClbuffer pH 1.2; (b) Acetate buffer pH 4.5; (c) Phosphate buffer pH 6.8;and (d) Phosphate buffer 7.4. See Example 1.

FIG. 7 exemplifies testosterone (T) solubilities following thedissolution of the following encapsulated formulations in PBS pH 6.80 at37° C.: F1 (60% T, 32% DSPC, and 8% Cholesterol); F2 (60% T, 36% DSPC,and 4% Cholesterol); F3 (60% T, 40% DSPC, and 0% Cholesterol); F4 (50%T, 40% DSPC, and 10% Cholesterol); F5 (50% T, 45% DSPC, and 5%Cholesterol); F6 (50% T, 50% DSPC, and 0% Cholesterol); F7 (40% T, 48%DSPC, and 12% Cholesterol); F8 (40% T, 54% DSPC, and 9% Cholesterol);and F9 (40% T, 60% DSPC, and 0% Cholesterol). See Example 1 and Table 4.

FIG. 8 exemplifies testosterone solubilities following the dissolutionof: (a) unformulated testosterone in 0.5% SLS; (b) unformulatedtestosterone in 1% SLS; (c) unformulated testosterone in 2% SLS; (d)PLF-2 in 0.5% SLS; (e) PLF-2 in 1% SLS; and (f) PLF-2 in 2% SLS. SeeExample 3.

FIG. 9 exemplifies the in vitro testosterone dissolution profiles ofencapsulated and enterically-coated PLF-1 that: (a) had not been stored;(b) stored for three months at room temperature; or (c) stored for threemonths at 30° C. See Example 4.

FIG. 10 exemplifies the mean plasma testosterone concentrations overtime following the oral administration of: (a) 300 mg/kg(testosterone/rat weight) of unformulated testosterone; (b) PLF-2comprising 300 mg/kg of testosterone; (c) PLF-2 comprising 150 mg/kg oftestosterone; and (d) PLF-4 comprising 300 mg/kg of testosterone to malefasted rats. See Example 5.

FIG. 11 exemplifies the mean plasma testosterone concentrations overtime following the oral administration of: a) 300 mg/kg(testosterone/rat weight) of unformulated testosterone; (b) 31 mg/kg(testosterone/rat weight) of unformulated testosterone; (c) PLF-2comprising 31 mg/kg of testosterone; (d) PLF-2 comprising 15.5 mg/kg oftestosterone; and (e) PLF-2 comprising 7.75 mg/kg of testosterone malefasted rats. See Example 5.

FIG. 12 exemplifies the in vitro testosterone dissolution profiles ofthe following formulations: (a) encapsulated PLF-5 with Avicel® PH 101and 1% Explotab® disintegrant; (b) tableted PLF-5 with Avicel® PH 101,SD lactose, Mg stearate; and 1% Explotab® disintegrant; (c) tabletedPLF-5 with Avicel® PH 101, Mg stearate; and 5% Explotab® disintegrant;and (d) powdered PLF-5 with Avicel® PH 101, Mg stearate; and 5%Explotab® disintegrant. See Example 6.

FIG. 13 exemplifies the in vitro testosterone dissolution profiles ofthe following tableted formulations: (a) PLF-6 with Avicel® PH 101, Mgstearate; and Explotab® disintegrant; (b) PLF-6 with 20% Pearlitol®,Avicel® PH 102, Mg stearate; and Explotab® disintegrant; (c) PLF-6 with10% Pearlitol®, Avicel® PH 102, Mg stearate; and Explotab® disintegrant;(d) PLF-7 with Avicel® PH 102, Mg stearate; and Explotab® disintegrant;and (e) PLF-6 with Avicel® PH 101, Mg stearate; and Explotab®disintegrant. Dissolution in (e) was performed in a Type II apparatus at75 rpm, rather then 50 rpm, as was done for (a)-(d). See Example 7.

FIG. 14 exemplifies the in vitro testosterone dissolution profiles ofthe following formulations: (a) encapsulated PLF-2 with Avicel® PH 101;(b) tableted PLF-6 with Avicel® PH 101, Mg stearate; and Explotab®disintegrant; and (c) PLF-8 (unformulated testosterone control). SeeExample 8 and Table 9.

FIG. 15 exemplifies the in vitro testosterone dissolution profiles ofthe following encapsulated formulations: (a) PLF-9 with Avicel® PH 102(1:2); (b) PLF-9 with Avicel® PH 102 (1:0.5); (c) PLF-9 with Avicel® PH102 (1:1); (d) PLF-9 with Avicel® PH 102 (1:0.6); and (e) PLF-9 withAvicel® PH 102 (1:0.6) and Explotab® disintegrant. See Example 9 andTable 10.

FIG. 16 exemplifies the in vitro testosterone dissolution profiles ofthe following encapsulated formulations and percentages of SLS in thedissolution media: (a) PLF-11 with Avicel® PH 102 dissolved in 1% SLS;(b) PLF-11 with Avicel® PH 102 dissolved in 2% SLS; (c) PLF-12 withAvicel® PH 102 and Explotab® disintegrant dissolved in 0.5% SLS; and (d)PLF-12 with Avicel® PH 102 and Explotab® disintegrant dissolved in 1%SLS. See Example 10 and Table 11.

FIG. 17 exemplifies the in vitro testosterone dissolution profiles ofthe following encapsulated formulations: (a) PLF-16 with Avicel® PH 102and Explotab® disintegrant; (b) PLF-17 with Avicel® PH 102 and Explotab®disintegrant; (c) PLF-18 with SMCC and Explotab® disintegrant; (d)PLF-19 with DCP and Explotab® disintegrant; (e) PLF-20 with Avicel® PH102 and Explotab® disintegrant; (f) PLF-21 with SMCC and Explotab®disintegrant; and (g) PLF-22 with DCP and Explotab® disintegrant. SeeExample 12, and Table 13.

FIG. 18 exemplifies the in vitro testosterone dissolution profiles ofencapsulated (a) PLF 24 (placebo) enteric coated and (b) entericallycoated PLF 25 containing testosterone. See Example 13 and Table 14.

FIG. 19 exemplifies the in vitro testosterone dissolution profiles ofthe following encapsulated formulations: (a) enterically-coated PLF-26;(b) enterically-coated PLF-27; and (c) uncoated PLF-29. See Example 14and Table 18.

FIG. 20 exemplifies the transport of testosterone released from theformulations listed in Table 20 across a monolayer of Caco-2 Cells. SeeExample 15B and Table 20.

FIG. 21 exemplifies the mean plasma concentrations of testosterone overtime obtained from human patients with hypogonadism following the oraladministration of either 120 mg or 240 mg of testosterone in humans asdescribed in the pharmacokinetics study of Example 16. Testosterone (T)concentrations are adjusted for baseline. Plasma testosteroneconcentrations were determined at the times shown after administrationof: (a) 120 mg of T under fasting conditions; (b) 240 mg of T underfasting conditions; (c) 120 mg of T under fed conditions; and (d) 240 mgof T under fed conditions.

FIG. 22 exemplifies the mean plasma concentrations of the testosteronemetabolite DHT over time obtained from human patients with hypogonadismfollowing the oral administration of either 120 mg or 240 mg oftestosterone that has been formulated as provided in Table 20. DHTconcentrations are adjusted for baseline. Plasma DHT concentrations weredetermined at the times shown after administration of either: (a) 120 mgof formulated T under fasting conditions; (b) 240 mg of formulated Tunder fasting conditions; (c) 120 mg of formulated T under fedconditions; and (d) 240 mg of formulated T under fed conditions.

FIG. 23 exemplifies mean plasma testosterone concentrations (24 hprofile) in human subjects with hypogonadism after receivingtwice-daily, oral administrations of either 120 mg or 240 mg offormulated testosterone (T) for a period of time of one day (D1) andfifteen days (D15). The 120 mg dosage form used to obtain the data isdescribed in Example 18. The 240 mg doses were given by administeringtwo 120 mg doses.

FIG. 24 exemplifies mean plasma testosterone (T) concentrations of humansubjects with hypogonadism every two hours over the first 24 hours of a15 day long treatment regimen of being administered a 120 mgtestosterone dosage form like the dosage form that is described inExample 18, herein, twice-daily.

FIG. 25 exemplifies mean plasma testosterone (T) concentrations of humansubjects with hypogonadism every two hours over the first 24 hours of a15 day long treatment regimen of being administered the 240 mgtestosterone proliposomal dosage form that is described in Example 18,herein, twice-daily.

FIG. 26 exemplifies the mean plasma testosterone concentrations inbeagle dogs over the 24 hour period of the first day of after orallyadministering: (a) placebo, (b) 120 mg, (c) 600 mg, or (d) 1200 mg oftestosterone that had been formulated according to the composition andmethod of preparation of the proliposomal testosterone dosage formdescribed in Example 18.

FIG. 27 exemplifies the mean plasma testosterone concentrations inbeagle dogs over a 24 hour period of the 57^(th) day after orallyadministering (a) placebo, (b) 120 mg, (c) 600 mg, or (d) 1200 mg oftestosterone that had been formulated according to the composition andmethod of preparation of the proliposomal testosterone dosage formdescribed in Example 18.

FIG. 28 exemplifies the mean plasma testosterone concentrations inbeagle dogs over a 24 hour period of the 91^(st) day after orallyadministering (a) placebo, (b) 120 mg, (c) 600 mg, or (d) 1200 mg oftestosterone that had been formulated according to the composition andmethod of preparation of the proliposomal testosterone dosage formdescribed in Example 18.

DETAILED DESCRIPTION Testosterone and Testosterone Deficiency

Disclosed herein, in certain embodiments, are compositions and methodsof treating testosterone deficiency. Further disclosed herein, incertain embodiments, are methods and compositions for treating diseases,disorders, or conditions characterized by testosterone deficiency. Anumber of situations (including aging, the use of androgen depletiontherapy for the treatment of prostate cancer, or genetic abnormalities)may result in abnormally low levels of testosterone (i.e., testosteronedeficiency). The consequences associated with testosterone deficiency inmen include, but are not limited to, increased fat mass, decreasedmuscle mass and strength, sexual dysfunction, and osteoporosis.Testosterone deficiency may also be associated with the development of avariety of metabolic and cardiovascular conditions.

As a man ages, the amount of testosterone in his body graduallydeclines. This natural decline starts after age 30 and continuesthroughout life. Other causes of low testosterone levels include, butare not limited to: injury, infection, or loss of the testicles;chemotherapy or radiation treatment for cancer; genetic abnormalitiessuch as Klinefelter's Syndrome (extra X chromosome); hemochromatosis(too much iron in the body); dysfunction of the pituitary gland (a glandin the brain that produces many important hormones); inflammatorydiseases such as sarcoidosis (a condition that causes inflammation ofthe lungs); medications, especially hormones used to treat prostatecancer and corticosteroid drugs; chronic illness; chronic kidneyfailure; liver cirrhosis; stress; alcoholism; obesity (especiallyabdominal); and congenital conditions, such as Kallman's Syndrome.

Male hypogonadism, or testosterone deficiency syndrome (TDS), resultsfrom a failure of the testes to produce adequate androgen. Patients havelow circulating testosterone in combination with clinical symptoms suchas fatigue, erectile dysfunction, and body composition changes. Thecause may be primary (genetic anomaly, Klinefelter's syndrome) orsecondary (defect in hypothalamus or pituitary), but often presents withthe same symptomatology. In the older patient, androgen deficiency ofthe aging male (ADAM) is an important cause of secondary hypogonadismbecause testosterone levels decline progressively after age 40.Hypogonadal patients have alterations not only in sexual function andbody composition, but also in cognition and metabolism. Regardless ofetiology, hypogonadal patients who are both symptomatic and who haveclinically significant alterations in laboratory values are candidatesfor treatment. The goal of hormone replacement therapy in these men isto restore hormone levels to the normal range and to alleviate symptomssuggestive of hormone deficiency. This can be accomplished in a varietyof ways, although most commonly testosterone replacement therapy (TRT)is employed. Disclosed herein, in certain embodiments, are proliposomalpowder dispersions for treating hypogonadism comprising: (a)testosterone, (b) cholesterol, and (c) at least one phospholipid,wherein (a) and (b) are present in a weight ratio (a):(b) ranging from1:0.05 to 1:0.30 and (a), (b) and (c) are present in a weight ratio of(a):((b)+(c)) ranging from 1:1.0 and 1:2.5. Further, disclosed herein,in certain embodiments, are methods of treating hypogonadism in anindividual in need thereof comprising administering to the individual aproliposomal powder dispersion comprising: (a) testosterone, (b)cholesterol, and (c) at least one phospholipid, wherein (a) and (b) arepresent in a weight ratio (a):(b) ranging from 1:0.05 to 1:0.30 and (a),(b) and (c) are present in a weight ratio of (a):((b)+(c)) ranging from1:1.0 and 1:2.5.

Klinefelter's syndrome, (47XXY, or XXY syndrome) is a condition in whichhuman males have an extra X chromosome. In humans, 47XXY is the mostcommon sex chromosome aneuploidy in males and the second most commoncondition caused by the presence of extra chromosomes. The physicaltraits of the syndrome become more apparent after the onset of puberty,if at all. Principal effects include hypogonadism and reduced fertility.A variety of other physical and behavioural differences and problems arecommon, though severity varies and many XXY boys have few detectablesymptoms. Not all XXY boys and men develop the symptoms of Klinefeltersyndrome. The genetic variation is irreversible. Testosterone treatmentis an option for some individuals who desire a more masculine appearanceand identity. Disclosed herein, in certain embodiments, are proliposomalpowder dispersions for treating Klinefelter's syndrome comprising: (a)testosterone, (b) cholesterol, and (c) at least one phospholipid,wherein (a) and (b) are present in a weight ratio (a):(b) ranging from1:0.05 to 1:0.30 and (a), (b) and (c) are present in a weight ratio of(a):((b)+(c)) ranging from 1:1 and 1:2.5. Further, disclosed herein, incertain embodiments, are methods of treating Klinefelter's syndrome inan individual in need thereof comprising administering to the individuala proliposomal powder dispersion comprising: (a) testosterone, (b)cholesterol, and (c) at least one phospholipid, wherein (a) and (b) arepresent in a weight ratio (a):(b) ranging from 1:0.05 to 1:0.30 and (a),(b) and (c) are present in a weight ratio of (a):((b)+(c)) ranging from1:1.0 and 1:2.5.

Other diseases or conditions where the level of endogenous testosteroneis insufficient include, but are not limited to, erectile dysfunction,idiopathic gonadotropin deficiency, pitutary hypothalamus injury due totumours, osteoporosis, diabetes mellitus, chronic heart failure,chemotherapy, hemochromatosis, cirrhosis, renal failure, AIDS,sarcoidosis, Kallman's Syndrome, androgen receptor defects, 5-alphareductase deficiency, myotonic dystrophy, cryptorchidism, mumpsorchitis, aging, fertile eunuch syndrome, and pituitary disorders.Disclosed herein, in certain embodiments, are proliposomal powderdispersions for treating diseases, disorders or conditions where thelevel of endogenous testosterone is insufficient comprising: (a)testosterone, (b) cholesterol, and (c) at least one phospholipid,wherein (a) and (b) are present in a weight ratio (a):(b) ranging from1:0.05 to 1:0.30 and (a), (b) and (c) are present in a weight ratio of(a):((b)+(c)) ranging from 1:1 and 1:2.5. Further, disclosed herein, incertain embodiments, are methods of treating diseases, disorders orconditions where the level of endogenous testosterone is insufficientcomprising administering to an individual in need thereof a proliposomalpowder dispersion comprising: (a) testosterone, (b) cholesterol, and (c)at least one phospholipid, wherein (a) and (b) are present in a weightratio (a):(b) ranging from 1:0.05 to 1:0.30 and (a), (b) and (c) arepresent in a weight ratio of (a):((b)+(c)) ranging from 1:1 and 1:2.5.In embodiments of any of the aforementioned, the disease, disorder orcondition where the level of endogenous testosterone is insufficient iserectile dysfunction, idiopathic gonadotropin deficiency, pitutaryhypothalamus injury due to tumours, osteoporosis, diabetes mellitus,chronic heart failure, chemotherapy, hemochromatosis, cirrhosis, renalfailure, AIDS, sarcoidosis, Kallman's Syndrome, androgen receptordefects, 5-alpha reductase deficiency, myotonic dystrophy,cryptorchidism, mumps orchitis, aging, fertile eunuch syndrome, andpituitary disorders. In embodiments of any of the aforementioned, thedisease, disorder or condition where the level of endogenoustestosterone is insufficient is erectile dysfunction. In embodiments ofany of the aforementioned, the disease, disorder or condition where thelevel of endogenous testosterone is insufficient is diabetes mellitus.In embodiments of any of the aforementioned, the disease, disorder orcondition where the level of endogenous testosterone is insufficient ischronic heart failure.

The therapeutic effectiveness of a drug depends on its bioavailability,i.e., the measure of the rate and extent to which the drug or activemoiety is absorbed from a drug product and becomes available at the siteof action. Poor bioavailability of a drug depends on many factors,particularly important factors include drug solubility in thegastro-intestinal fluid (GI fluid), drug stability in the GI region(acid and enzyme stability), and systemic concentration of the drugwithout significant loss to the hepatic portal system before reachingthe rest of the body (the first pass effect). If a drug fails in one ofthese aspects, the drug may not be sufficiently available for biologicalactivity.

The FDA's Biopharmaceutics Classification System (BCS) provides guidancefor predicting oral drug absorption by taking into account a drug'saqueous solubility and its tissue permeability. Specifically, the BCSdivides drugs into classes I through IV based on their aqueoussolubility and permeability. Class I drugs are highly soluble and highlypermeable, class II drugs have low solubility, but are highly permeable,class III drugs are highly soluble but poorly permeable; and class IVdrugs are low soluble and poorly permeable.

Testosterone is a BCS class II drug. Unless otherwise indicated, theterm “testosterone” means testosterone that is chemically identical totestosterone produced by the human body, i.e, “native,” or “endogenous”testosterone, or a testosterone derivative. Testosterone may be isolatedform natural sources or made by commercial synthetic processes. In someembodiments, the testosterone used in the proliposomal powderdispersions described herein is testosterone that is chemicallyidentical to testosterone produced by the human body, i.e, “native,” or“endogenous” testosterone, or a testosterone derivative. In someembodiments, the testosterone used in the proliposomal powderdispersions described herein is a testosterone derivative, wherein theamount of a testosterone derivative in the proliposomal dispersioncorresponds to a molar amount of testosterone allowed in a proliposomaldispersion based on the range of weight: weight ratios of testosteroneto the other proliposomal dispersion components that are specifiedherein. In some embodiments, the testosterone used in the proliposomalpowder dispersions described herein is a testosterone derivativeselected from: testosterone undecanoate (or testosterone undecylate),epitestosterone, fluoxymesterone, mesterolone, methyltestosterone,19-nortestosterone, 17-alpha methyl testosterone, 7-alpha alkyl19-nortestosterone, or testosterone enanthate. In some embodiment, thetestosterone used in the pharmaceutical compositions described herein isesterified at the 17-beta hydroxyl. In some embodiments, esterificationof 17-beta hydroxyl group increases hydrophobicity. In some embodiment,the testosterone used in the proliposomal powder dispersions describedherein is a salt of testosterone. Examples of testosterone saltsinclude, but are not limited to: testosterone acetate and testosteronepropionate. In some embodiments, a proliposomal powder dispersiondescribed herein comprises testosterone that is chemically identical totestosterone produced by the human body, i.e, “native,” or “endogenous”testosterone, a testosterone derivative or testosterone salt, orindividual combinations thereof.

A major obstacle to successful commercialization of testosterone is thedifficulty of enhancing not only its dissolution rate but also theextent of its dissolution. Indeed, the fact that BCS class II drugs,like testosterone, are poorly soluble is often associated with low andhighly variable bioavailabilities of these drugs. Another drawback ofadministering testosterone is that much of ingested testosterone ismetabolized before it reaches its target(s). More specifically, thehepatic portal system that carries testosterone from the intestine tothe liver where it is metabolized before reaching systemic circulation.The so-called name given to this phenomenon is “the first pass effect.”In addition to the fact that the metabolized drug does not provide thesame effect as that of the parent drug, the first pass effect may alsocause hepato-toxicity. As discussed below, different approaches havebeen developed in order to avoid the first pass effect that isassociated with administering testosterone.

One approach to avoiding the first pass effect during testosteronetreatment has been to use prodrug forms of testosterone and othertestosterone derivatives that are not subject to the first pass effect.Indeed, ester prodrugs of testosterone, such as, testosteronepropionate, testosterone cypionate, testosterone enanthate, testosteronedecanoate, and testosterone undecanoate have been used as substitutesfor testosterone, e.g., sold under the Restandol® and Andriol®tradenames. In the United States, another testosterone derivative iscurrently available in different dosage forms: testosterone enanthatefor intramuscular injection (under the Delatestryl® tradename), topicalsolution (under the Axiron® tradename), buccal patch (under the Striant®tradename), topical gel (under the AndroGel® and AndroGel® 1.62%tradenames).

Another approach to testosterone treatment is based on thewell-established observation that circulating testosterone in a healthymale is metabolized to dihydrotestosterone (DHT) and to estradiol.Typically, the ratio of testosterone to DHT is 10:1 and to that toestradiol is 200:1. Changes in these ratios affect androgenic activity.As such, testosterone derivatives that restore the ratio of testosteroneto its metabolites have been used clinically. These types oftestosterone derivatives, such as alkylated derivatives used for oraltherapy (methyltestosterone), are metabolized slowly, thus allowinghigher levels of DHT, which, in turn, change the circulatingtestosterone: DHT ratios. However, prolonged use of alkylatedtestosterone derivatives has been associated with development of severehepatotoxicity.

Yet another approach to testosterone treatment is the utilization ofliposomes as effective oral carrier systems for testosterone. Liposomesabsorbed by the intestinal lymphatic system are incorporated intochylomicrons (i.e., a type of lipoprotein particle formed in theabsorptive cells of the small intestine). The chylomicrons, togetherwith remaining liposomes, bypass the liver (portal circulation), andtravel through the lymph system to the subclavian vein. In the bloodstream, these liposomes are digested by lipoprotein lipases derived fromthe capillary walls, which releases the drug. However, in spite of theadvantages liposome drug delivery offer, they also face obstaclespresented by the gastrointestinal digestive fluids, pH variation, bilesalts and lipolytic and proteolytic enzymes. For example, typicalliposomes are mostly broken down by the gastric acids, rendering thedelivery system ineffective for oral administration.

One approach to liposome-mediated drug delivery that offers theadvantages of liposomes, but minimizes the destructive effect of thegastrointestinal system on liposomes, is to use a proliposomal drugdelivery system. By employing this approach, a poorly-water soluble drugsuch as testosterone can be incorporated into a dry, free-flowing powderthat will form liposome-encapsulated testosterone in an aqueousenvironment. Because proliposomal formulation are dry powders, they,unlike liposomes, can be coated with a delayed release coating (e.g., anenteric coating)” that will protect the formulation until it reaches theless hostile environment of the small intestine, where liposomes canform in a dramatically less destructive environment. A need exists todevelop proliposomal pharmaceutical formulations that increase thesolubilities of testosterone and have it available at the site ofaction.

In some embodiments, the proliposomal powder dispersions and thepharmaceutical compositions disclosed herein are administered to anindividual in need of testosterone replacement therapy. In someembodiments, the proliposomal powder dispersions and the pharmaceuticalcompositions disclosed herein are administered to an individual in needthereof to treat erectile dysfunction, hypogonadism, idiopathicgonadotropin deficiency, pitutary hypothalamus injury due to tumours,osteoporosis, diabetes mellitus, chronic heart failure, chemotherapy,Klinefelter's Syndrome, hemochromatosis, cirrhosis, renal failure, AIDS,sarcoidosis, Kallman's Syndrome, androgen receptor defects, 5-alphareductase deficiency, myotonic dystrophy, cryptorchidism, mumpsorchitis, aging, fertile eunuch syndrome, pituitary disorders, and otherconditions where the level of endogenous testosterone is insufficient.Accordingly, disclosed herein, in certain embodiments, are methods oftreating an individual in need of testosterone therapy comprisingadministering to the individual a therapeutically effective amount ofthe proliposomal powder dispersion described herein. In someembodiments, the proliposomal powder dispersion or pharmaceuticalcomposition is administered orally and in a dosage form describedherein.

Pharmaceutical Compositions

The pharmaceutical compositions described herein are formulated fordelivery by any suitable method. Exemplary forms of the pharmaceuticalcompositions described herein, include, a tablet, a pill, a powder, acapsule (including both soft or hard capsules made from animal-derivedgelatin or plant-derived HPMC), a sachet, a troche, pellets, granules,emulsions, and solutions. These pharmaceutical compositions describedherein can be manufactured by conventional techniques known in thepharmaceutical arts.

Conventional techniques for preparing pharmaceutical compositionsdescribed herein include, e.g., one or a combination of methods: (1) drymixing, (2) direct compression, (3) milling, (4) dry or non-aqueousgranulation, (5) wet granulation, or (6) fusion. See, e.g., Lachman etal., The Theory and Practice of Industrial Pharmacy (1986). Othermethods include, e.g., prilling, spray drying, pan coating, meltgranulation, granulation, wurster coating, tangential coating, topspraying, tableting, extruding, coacervation and the like.

Disclosed herein, in certain embodiments, are novel testosteroneformulations where a pharmaceutically effective amount of testosteroneis incorporated into a phospholipid/cholesterol system to produce aproliposomal powder dispersion. In some embodiments, the proliposomalpowder dispersions are contained in delayed-release capsule for oraladministration and to withstand the acidic environment in the stomach.In some embodiments, the proliposomal powder dispersions are containedin an enterically-coated capsule for oral administration and towithstand the acidic environment in the stomach. Upon contact with smallintestinal fluid, the proliposomal powder dispersion is dispersed andhydrated, leading to the formation of liposomes and uptake of thetestosterone through the lymphatic system. In some embodiments, thecompositions disclosed herein reduce the first-pass side effect that iscommonly associated with oral administration of testosterone.

In some embodiments, a proliposomal powder dispersion disclosed hereincomprises (a) testosterone, (b) cholesterol, and (c) at least onephospholipid. As used herein, a “proliposomal powder dispersion” means amixture of at least (a) testosterone, (b) cholesterol, and (c) at leastone phospholipid, dispersed one in another, and which forms a liposomeupon contact with an aqueous environment. As shown in examples below,proliposomal powder dispersions with low amounts of cholesterol possessimproved permeability with less standard deviation as compared to powderdispersions with no cholesterol or powder dispersions containingrelatively large amounts of cholesterol.

In some embodiments, testosterone (a) and cholesterol (b) are present ina weight ratio of (a):(b) that ranges from about 1:about 0.05 to about1:about 0.3. In some embodiments, testosterone (a) and cholesterol (b)are present in a weight ratio of (a):(b) that ranges from 1:0.05 to1:0.3. In some embodiments, testosterone (a) and cholesterol (b) arepresent in a weight ratio of (a):(b) is about 1:about 0.05, about1:about 0.1, about 1:about 0.15, about 1:about 0.2, about 1:about 0.25,or about 1:about 0.3. In some embodiments, testosterone (a) andcholesterol (b) are present in a weight ratio of (a):(b) is 1:0.05,1:0.1, 1:0.15, 1:0.2, 1:0.25, or 1:0.3. In some embodiments, the ratioof testosterone to (b) and the at least one phospholipid (c), i.e.,(a):((b)+(c)) range from about 1:about 1 to about 1:about 2.5. In someembodiments, the ratio of testosterone to (b) and the at least onephospholipid (c), i.e., (a):((b) (c)) range from 1:1 to 1:2.5. In someembodiments, the ratio of testosterone to (b) and the at least onephospholipid (c), i.e., (a):((b)+(c)) is about 1:about 1, about 1:about1.1, about 1:about 1.2, about 1:about 1.3, about 1:about 1.4, about1:about 1.5, about 1:about 1.6, about 1:about 1.7, about 1:about 1.8,about 1:about 1.9, about 1:about 2.0, about 1:about 2.1, about 1:about2.2, about 1:about 2.3, about 1:about 2.4, or about 1:about 2.5. In someembodiments, the ratio of testosterone to (b) and the at least onephospholipid (c), i.e., (a):((b)+(c)) is 1:1, 1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3,1:2.4, or 1:2.5. The weight ratios of the components in the powderdispersion may be varied within these ranges. In some embodiments, theweight ratios of testosterone:cholesterol: at least one phospholipidare: about 1:about 0.1:about 0.9; about 1:about 0.2:about 1.8; and about1:about 0.2:about 1.3. In some embodiments, the weight ratios oftestosterone:cholesterol: at least one phospholipid are: 1:0.1:0.9;1:0.2:1.8; and 1:0.2:1.3. In certain embodiments, the weight ratio of(a):(b) ranges from about 1:about 0.1 to about 1:about 0.2 and (a), (b)and the at least one phospholipid (c) are present in a weight ratio of(a):((b)+(c)) ranging from about 1:about 1.1 and about 1:about 2. Incertain embodiments, the weight ratio of (a):(b) ranges from 1:0.1 to1:0.2 and (a), (b) and the at least one phospholipid (c) are present ina weight ratio of (a):((b)+(c)) ranging from 1:1.1 and 1:2.

In some embodiments, a proliposomal powder dispersion disclosed hereincomprises (a) testosterone, (b) cholesterol, and (c) at least onephospholipid. In some embodiments, a proliposomal powder dispersiondisclosed herein comprises (a) testosterone, (b) cholesterol, and (c)two phospholipids. The phospholipid component of a powder dispersiondisclosed herein is any pharmaceutically acceptable phospholipid andmixtures of such phospholipids. Natural as well as syntheticphospholipids may be used. Phospholipids are molecules that have twoprimary regions, a hydrophilic head region comprised of a phosphate ofan organic molecule and one or more hydrophobic fatty acid tails.Naturally-occurring phospholipids generally have a hydrophilic regioncomprised of choline, glycerol and a phosphate and two hydrophobicregions comprised of fatty acid. When phospholipids are placed in anaqueous environment, the hydrophilic heads come together in a linearconfiguration with their hydrophobic tails aligned essentially parallelto one another. A second line of molecules then aligns tail-to-tail withthe first line as the hydrophobic tails attempt to avoid the aqueousenvironment. To achieve maximum avoidance of contact with the aqueousenvironment, i.e., at the edges of the bilayers, while at the same timeminimizing the surface area to volume ratio and thereby achieve aminimal energy conformation, the two lines of phospholipids, known as aphospholipid bilayer or a lamella, converge into a liposome. In doingso, the liposomes (or phospholipid spheres) entrap some of the aqueousmedium, and whatever may be dissolved or suspended in it, in the core ofthe sphere. This includes components of a proliposomal powder dispersiondisclosed herein, such as testosterone and other components.

Examples of suitable phospholipids that may be used in a proliposomalpowder dispersion disclosed herein include but are not limited todistearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine,dimyristoyl phosphatidylcholine, egg phosphatidylcholine, soyphosphatidylcholine, dimyristyl phosphatidyl glycerol sodium,1,2-dimyristoyl-phosphatidic acid, dipalmitoylphosphatidylglycerol,dipalmitoyl phosphate, 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol,1,2-distearoyl-sn-glycero-3-phosphatidic acid, phosphatidylserine andsphingomyelin. The proliposomal powder dispersions disclosed herein mayalso comprise individual combinations of any of the aforementionedphospholipids. In some embodiments, phospholipids containing saturatedfatty acids are employed. In certain instances, use of phospholipidscontaining saturated fatty acids avoid stability (chemical) problemssometimes associated with unsaturated fatty acids. The phospholipid,distearoyl phosphatidylcholine, has been found to be particularly usefulin the powder dispersions described herein. In some embodiments, thephospholipid is distearoyl phosphatidylcholine.

In some embodiments, a proliposomal powder dispersion disclosed hereincomprises (a) testosterone, (b) cholesterol, and (c) at least onephospholipid. In some embodiments, a proliposomal powder dispersiondisclosed herein comprises (a) testosterone, (b) a cholesterolderivative, and (c) at least one phospholipid. In some embodiments, aproliposomal powder dispersion disclosed herein comprises (a)testosterone, (b) cholesterol, a cholesterol derivative, or acombination thereof, and (c) at least one phospholipid, wherein theamount of a cholesterol derivative in the proliposomal dispersioncorresponds to a molar amount of cholesterol allowed in a proliposomaldispersion based on the range of weight: weight ratios of cholesterol tothe other proliposomal dispersion components that are specified herein.The cholesterol component of the proliposomal powder dispersiondisclosed herein is either cholesterol (3β-hydroxy-5-cholestene5-cholesten-3β-ol) or a cholesterol derivative. Examples of suitablecholesterol and derivatives include, but are not limited to:22(R)-hydroxycholesterol; 22(S)-hydroxycholesterol;25-hydroxycholesterol; 5-cholesten-3β-ol-7-one; 5α-cholest-7-en-3β-ol;5α-cholestan-3β-ol; 5α-cholestane; 5β-cholestan-3α-ol;7β-hydroxycholesterol; campesterol; cholesta-3,5-diene; cholestanol;cholesterol 5α,6α-epoxide; cholesterol 5β,6β-epoxide; cholesterol-PEG600; cholesterol; cholesteryl 10-undecenoate; cholesteryl acetate;cholesteryl arachidonate; cholesteryl behenate; cholesteryl butyrate;cholesteryl caprylate; cholesteryl dodecanoate; cholesteryl elaidate;cholesteryl erucate; cholesteryl heptadecanoate; cholesteryl heptanoate;cholesteryl hexanoate; cholesteryl linoleate; cholesteryl linolelaidate;cholesteryl myristate; cholesteryl N-(trimethylammonioethyl)carbamatechloride; cholesteryl n-decanoate; cholesteryl n-valerate; cholesterylnervonate; cholesteryl oleate; cholesteryl palmitate; cholesterylpalmitelaidate; cholesteryl pelargonate; cholesteryl phenylacetate;cholesteryl stearate; glycocholic acid hydrate; lanosterol; sodiumcholesteryl sulfate; stigmastanol; SyntheChol®; Thiocholesterol. Theproliposomal powder dispersions disclosed herein may also compriseindividual combinations of any of the aforementioned cholesterolcomponents.

In some embodiments, a proliposomal powder dispersion disclosed hereinis prepared by first dissolving testosterone in a solvent. The solventis any solvent in which testosterone dissolves, but is preferably awater-miscible solvent. Examples of such solvents include, but are notlimited to, ethanol, methanol, chloroform, dichloromethane, acetone,isopropyl alcohol, and diethyl ether. If water is to be present incombination with a water-miscible solvent, (e.g., an ethanol and watersolvent system), the water may be added before or after the testosteroneis dissolved. Solvent-water systems are also made on a volume to volumebasis, and the amount of water should not be so much that the phospholidforms liposomes. Generally, the solvent solution should not comprise 10%or more of water (vol/vol). Once the testosterone is dissolved, in someembodiments cholesterol, and at least one phospholipid, as well as anyother proliposomal powder components are added to the solution and mixedto form a solution or dispersion of the components in the solvent. Invarious embodiments, the amounts of phospholipid and cholesterol addedto the solution are such that the ratio (wt/wt) of testosterone tophospholipid to cholesterol ranges from(0.50-3.50):(0.50-3.00):(0.05-0.50). In various embodiments, the amountsof phospholipid and cholesterol added to the solution are such that theratio (wt/wt) of testosterone to phospholipid to cholesterol range from(about 0.50-about 3.50):(about 0.50-about 3.00):(about 0.05-about 0.50).In some embodiments, the ratio (wt/wt) of testosterone to phospholipidto cholesterol ranges: (1.00:0.90:0.10); (1.50:1.35:0.15);(1.50:2.70:0.30); (1.00:1.35:0.15); or (3.00:2.70:0.30), respectively,as well as any ratio in between these ratios. In some embodiments, thethe ratio (wt/wt) of testosterone to phospholipid to cholesterol ranges:(about 1.00:about 0.90:about 0.10); (about 1.50:about 1.35:about 0.15);(about 1.50:about 2.70:about 0.30); (about 1.00:about 1.35:about 0.15);or (about 3.00:about 2.70:about 0.30), respectively, as well as anyratio in between these ratios. The dissolution of testosterone and themixing of other components is done in one or a series of steps and byany suitable means and preferably by stirring. In some embodiments,after mixing, the solvent is removed to yield a powder. The solvent isremoved by suitable techinique, for example, by evaporation, by placingthe solution under vacuum, by spray-drying, or by use of a drying gas,and the like. In some embodiments, the components are mixed by stirringat room temperature until the solvent evaporates, i.e., by stirringovernight. In some embodiments, a method of removing the solvent furthercomprises use of heat. The particle size of resulting powder dispersionmay be reduced by grinding, passing the powder through screens, or byany other suitable technique. In some embodiments, the particles withina proliposomal powder dispersion described herein may have powder sizeranging from about 10 to 200 mesh, 20 to 120 mesh or 40 to 80 mesh. Ifdesired, the proliposomal powder disperison may undergo further dryingto remove or reduce the amount of any residual solvent still present inthe powder. Such a further drying step is performed by using one or moreof the drying techniques discussed above or by other suitable dryingtechnique.

In some embodiments, a proliposomal powder dispersion disclosed hereinfurther comprises other pharmaceutically acceptable excipients. Whenpreparing a proliposomal powder dispersion described herein, excipientsare generally added to the combined, powdered mixture of testosterone,cholesterol and phospholipid, i.e., excipients are added “externally.”For example, the free-flowing powdered formulation may be admixed withat least one pharmaceutically acceptable excipient. Exemplarypharmaceutically acceptable excipients include, but are not limited to:(a) fillers or extenders, such as, for example, starches, lactose (e.g.,lactose monohydrate), sucrose, glucose, mannitol, and silicic acid; (b)binders, such as, for example, cellulose derivatives, includingmicrocrystalline cellulose, e.g., the various Avicel® PH products (FMCBioPolymer—Philadelphia, Pa.) (e.g., Avicel® PH-101 and PH-102), andProsolv® SMCC 90 and Prosolv® SMCC 90 HD (JRS Pharma—Rosenberg,Germany), starch, aliginates, gelatin, polyvinylpyrrolidone, sucrose,and gum acacia, (c) humectants, such as, for example, glycerol, (d)disintegrating agents, such as, for example, agar-agar, calciumcarbonate, potato or tapioca starch, e.g., sodium starch glycolate(e.g., Explotab® disintegrant (JRS Pharma—Rosenberg, Germany)), alginicacid, croscarmellose sodium, complex silicates, and sodium carbonate,(e) solution retarders, such as, for example, paraffin, (f) absorptionaccelerators, such as, for example, quaternary ammonium compounds, (g)wetting agents, such as, for example, cetyl alcohol, and glycerolmonostearate, magnesium stearate and the like, (h) adsorbents, such as,for example, kaolin and bentonite, (i) lubricants, such as, for example,talc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate (SLS), (j) plasticizers, and (k) dispersants,including mannitol, e.g., Pearlitol® SD 2000 (Roguette Pharma—Lestrem,France), or combinations thereof. In some embodiments, thepharmaceutically acceptable excipients are used in any suitable amountfor the particular excipient. Where the excipient is not a binder, insome embodiments the excipients are present in a testosterone: excipientweight ratio ranging from about 1:about 0.05 to about 1:about 0.3, andfrom about 1:about 0.1 to about 1:about 0.2. Where the excipient is nota binder, in some embodiments the excipients are present in atestosterone: excipient weight ratio ranging from 1:0.05 to 1:0.3, andfrom about 1:0.1 to 1:0.2. Where the excipients are binders, in someembodiments the testosterone: excipient weight ratio ranges from about1:about 0.5 to about 1:about 4. Where the excipients are binders, insome embodiments the testosterone: excipient weight ratio ranges from1:0.5 to 1:4.

In some embodiments, a formulation described herein further comprises adisintegrating agent. In various embodiments, the formulation comprisesthe disintegrating agent, sodium starch glycolate, e.g., Explotab®disintegrant. Other suitable disintregating agents may also be sued. Insome embodiments, the ratio of disintegrating agent to the combinedmixture of testosterone, phospholipid, and cholesterol (“theproliposomal powder component of the formulation”) ranges from about1:about 10 to about 1:about 35 (wt/wt), which correlates to a ratio ofdisintegrating agent to testosterone that ranges from about 1:about 5 toabout 1:about 20. In some embodiments, the ratio of disintegrating agentto the combined mixture of testosterone, phospholipid, and cholesterol(“the proliposomal powder component of the formulation”) ranges from1:10 to 1:35 (wt/wt), which correlates to a ratio of disintegratingagent to testosterone that ranges from 1:5 to 1:20.

In some embodiments, a formulation described herein further comprises abinder. Because binders are often classified according to particle size,the particle size of the binder excipient that is included in theformulation is selected based on the knowledge and skill of one of skillin the pharmaceutical formulation arts. In some embodiments, the binderis a microcrystalline cellulose, such as Avicel®PH-101, which has aparticle size of 50 μm, or Avicel® PH-102, which has a particle size of100 μm. Alternatively, in other embodiments, the binder is Prosolv® SMCC90 or Prosolv® SMCC 90 HD which are both microcrystalline cellulosebinders, each with a particle size of 110 μm. In still otherembodiments, the binder is dibasic calcium phosphate (DCP). In someembodiments, the ratio of microcrystalline cellulose binder to theproliposomal powder component of the formulation ranges from from 1:1 to3:1 (wt/wt), which correlates to a ratio of binder to testosterone thatranges from 4:1 to 1.5:1. In some embodiments, the ratio ofmicrocrystalline cellulose binder to the proliposomal powder componentof the formulation ranges from from about 1:about 1 to about 3:about 1(wt/wt), which correlates to a ratio of binder to testosterone thatranges from 4:1 to 1.5:1. In some embodiments, the binder is DCP and theratio of binder to the proliposomal powder component ranges from 0.25:1to 1.6:1 wt/wt), which correlates to a ratio of binder to testosteronethat ranges from 0.20:1 to 0.50:1. In some embodiments, the binder isDCP and the ratio of binder to the proliposomal powder component rangesfrom about 0.25:about 1 to about 1.6:about 1 wt/wt), which correlates toa ratio of binder to testosterone that ranges from about 0.20:about 1 toabout 0.50:about 1.

In some embodiments, a formulation described herein further comprises alubricant. In some embodiments, the formulation comprises the lubricant,magnesium (Mg) stearate. In some embodiments, the ratio of lubricant tothe proliposomal powder component of the formulation ranges from 145:1to 225:1 (wt/wt), which correlates to a ratio of lubricant totestosterone that ranges from 70:1 to 115:1. In some embodiments, theratio of lubricant to the proliposomal powder component of theformulation ranges from about 145:about 1 to about 225:about 1 (wt/wt),which correlates to a ratio of lubricant to testosterone that rangesfrom about 70:about 1 to about 115:about 1.

In some embodiments, a formulation described herein further comprises adispersant. In some embodiments, the formulation comprises thedispersant, mannitol, e.g., Pearlitol® SD 200. In some embodiments, theratio of dispersant to the proliposomal powder component of theformulation ranges from 0.20:1 to 0.60:1 (wt/wt), which correlates to aratio of dispersant to testosterone that ranges from 0.40:1.0 to1.5:1.0. In some embodiments, the ratio of dispersant to theproliposomal powder component of the formulation ranges from about0.20:about 1 to about 0.60:about 1 (wt/wt), which correlates to a ratioof dispersant to testosterone that ranges from about 0.40:about 1.0 toabout 1.5:about 1.0.

Disclosed herein, in certain embodiments, is an oral dosage formcomprising a proliposomal powder dispersion or a pharmaceuticalcomposition described herein. Exemplary forms of the pharmaceuticalcompositions described herein, include, a tablet, a pill, a powder, acapsule (including both soft or hard capsules made from animal-derivedgelatin or plant-derived HPMC), a sachet, a troche, pellets, granules,emulsions, and solutions. In some embodiments, the oral dosage form is atablet or a capsule. In some embodiments, the oral dosage form has adelayed release coating (e.g., an enteric coating). In some embodiments,the oral dosage form has an enteric coating.

Tablets may be prepared by any suitable technique (e.g., compressiontechniques). Conventional techniques include, e.g., one or a combinationof methods: (1) dry mixing, (2) direct compression, (3) milling, (4) dryor non-aqueous granulation, (5) wet granulation, or (6) fusion. See,e.g., Lachman et al., The Theory and Practice of Industrial Pharmacy(1986). Other methods include, e.g., spray drying, pan coating, meltgranulation, granulation, fluidized bed spray drying or coating (e.g.,wurster coating), tangential coating, top spraying, tableting, extrudingand the like.

Compressed tablets are solid dosage forms prepared by compacting thebulk blend formulations described above. In other embodiments, thecompressed tablets will include a film surrounding the final compressedtablet. In some embodiments, the film coating aids in patient compliance(e.g., Opadry® coatings or sugar coating). Film coatings comprisingOpadry® typically range from about 1% to about 5% of the tablet weight.In other embodiments, the compressed tablets include one or moreexcipients.

Provided herein are pharmaceutical compositions in film-coated dosageforms, which comprise a combination of an active ingredient, or apharmaceutically acceptable salt, solvate, or prodrug thereof; and oneor more tabletting excipients to form a tablet core using conventionaltabletting processes and subsequently coating the core. The tablet corescan be produced using conventional granulation methods, for example wetor dry granulation, with optional comminution of the granules and withsubsequent compression and coating.

In some embodiments, compressed tablets are solid dosage forms preparedby compacting the bulk blend compositions described above. In someembodiments, the compressed tablets comprise a film surrounding thefinal compressed tablet.

Further provided herein are pharmaceutical compositions in entericcoated dosage forms, which comprise a combination of an activeingredient, or a pharmaceutically acceptable salt, solvate, or prodrugthereof; and one or more release controlling excipients for use in anenteric coated dosage form. The pharmaceutical compositions alsocomprise non-release controlling excipients. Enteric-coatings arecoatings that resist the action of stomach acid but dissolve ordisintegrate in the intestine.

Capsules include both soft and hard capsules, e.g., capsules made fromanimal-derived gelatin or plant-derived HPMC. In some embodiments, thecapsule is a size 5, 4, 3, 2, 1, 0, 0E, 00, 000, 13, 12, 12el, 11, 10, 7or Su07. In various embodiments, the capsule is a size ‘00’ Vcaps or ahard gelatin capsule. In some embodiments, the capsules are filled withthe powdered proliposomal testosterone formulation disclosed herein,including excipients.

In one embodiment, pharmaceutical compositions which are used orallyinclude push-fit capsules made of gelatin, as well as soft, sealedcapsules made of gelatin and a plasticizer, such as glycerol orsorbitol. In addition, in one embodiment, stabilizers are added.

Capsules are filled using any suitable techniques. A capsule may beprepared by placing the bulk blend composition, described above, insidea capsule.

It should be understood that many carriers and excipients may serveseveral functions, even within the same formulation.

As discussed above, the filled capsules may be coated with a delayedrelease coating (e.g., an enteric coating). In some embodiments, thedelayed release coating (e.g., an enteric coating) releases theproliposomal powder dispersion in the small intestine. In variousembodiments, the delayed release coating (e.g., an enteric coating)composition comprises a polymer, such as an aqueous dispersion ofanionic polymers with methacrylic acid as a functional group (e.g.,Eudragit® L30D-55 (Evonik Industries). In some embodiments, the delayedrelease coating (e.g., an enteric coating) composition comprises aplasticizer (e.g., triethyl citrate). In some embodiments, the delayedrelease coating (e.g., an enteric coating) composition comprises ananti-tacking agent (e.g., talc). In some embodiments, the delayedrelease coating (e.g., an enteric coating) composition comprises adiluent such as water. In some embodiments, the coating compositioncomprises about: about 42 weight % (wt %) of an aqueous dispersion ofanionic polymers with methacrylic acid as a functional group; about 1.25wt % of a plasticizer; about 6.25 wt % of an anti-tacking agent; andabout 51 wt % of a diluent. In some embodiments, the coating compositioncomprises about: 42 weight % (wt %) of an aqueous dispersion of anionicpolymers with methacrylic acid as a functional group; 1.25 wt % of aplasticizer; 6.25 wt % of an anti-tacking agent; and 51 wt % of adiluent. In some embodiments, for example when a large-scalepreparations are preferred, an appropriate amount of an anioniccopolymer based on methacrylic acid and ethyl acrylate, such asEudragit® L100-55, is used in place of Eudragit® L30D-55. The coatingcomposition is applied to the capsules by using any suitable method,such as, but not limited to using a Procept pan coating machine andCaleva mini coater air suspension coating machine to coat the capsulesuntil they experience a 10% to 15% weight gain.

In some embodiments, the solid dosage forms described herein can beformulated as enteric coated delayed release oral dosage forms, i.e., asan oral dosage form of a pharmaceutical composition as described hereinwhich utilizes a delayed release coating (e.g., an enteric coating) toaffect release in the small intestine of the gastrointestinal tract.

In some embodiments, the solid dosage forms described herein are coated.In various embodiments contemplated herein, the coating is, for example,a gastric resistant coating such as an delayed release coating (e.g., anenteric coating), a controlled-release coating, an enzymatic-controlledcoating, a film coating, a sustained-release coating, animmediate-release coating, a delayed-release coating, or a moisturebarrier coating. See, e.g, Remington's Pharmaceutical Sciences, 20thEdition (2000).

In some embodiments a delayed release formulation is prepared by (a)spraying the proliposomal dispersion on to nonpareil beads by top sprayconfiguration, (b) coating the beads with a barrier coat, and (c)coating the beads an delayed release coating (e.g., an enteric coating)polymer. The enteric coated nonpareil beads are then formulated astablets or capsules.

The term “delayed release” as used herein refers to the delivery so thatthe release can be accomplished at some generally predictable locationin the intestinal tract more distal to that which would have beenaccomplished if there had been no delayed release alterations. In someembodiments, the pharmaceutical compositions described herein releasethe testosterone in the small intestine. In some embodiments, thepharmaceutical compositions described herein release the testosterone inthe duodenum, jejunum or ileum. In some embodiments, the pharmaceuticalcompositions described herein release the testosterone in the largeintestine. In some embodiments the method for delay of release iscoating. Any coatings should be applied to a sufficient thickness suchthat the entire coating does not dissolve in the gastrointestinal fluidsat pH below about 5, but does dissolve at pH about 5 and above. It isexpected that any anionic polymer exhibiting a pH-dependent solubilityprofile can be used as a delayed release coating (e.g., an entericcoating) in the methods and compositions described herein to achievedelivery to the lower gastrointestinal tract. In some embodiments thepolymers described herein are anionic carboxylic polymers. In otherembodiments, the polymers and compatible mixtures thereof, and some oftheir properties, include, but are not limited to:

(a) Shellac, also called purified lac, a refined product obtained fromthe resinous secretion of an insect. This coating dissolves in media ofpH>7;

(b) Acrylic polymers. The performance of acrylic polymers (primarilytheir solubility in biological fluids) can vary based on the degree andtype of substitution. Examples of suitable acrylic polymers includemethacrylic acid copolymers and ammonium methacrylate copolymers. TheEudragit® series E, L, S, RL, RS and NE (Evonik industries) areavailable as solubilized in organic solvent, aqueous dispersion, or drypowders. The Eudragit® series RL, NE, and RS are insoluble in thegastrointestinal tract but are permeable and are used primarily forcolonic targeting. The Eudragit® series E dissolve in the stomach. TheEudragit® series L, L-30D and S are insoluble in stomach and dissolve inthe intestine;

(c) Cellulose Derivatives. Examples of suitable cellulose derivativesare: ethyl cellulose; reaction mixtures of partial acetate esters ofcellulose with phthalic anhydride. The performance can vary based on thedegree and type of substitution. Cellulose acetate phthalate (CAP)dissolves in pH>6. Aquateric (FMC) is an aqueous based system and is aspray dried CAP psuedolatex with particles <1 μm. Other components inAquateric can include pluronics, Tweens, and acetylated monoglycerides.Other suitable cellulose derivatives include: cellulose acetatetrimellitate (Eastman); methylcellulose (Pharmacoat, Methocel);hydroxypropylmethyl cellulose phthalate (HPMCP); hydroxypropylmethylcellulose succinate (HPMCS); and hydroxypropylmethylcellulose acetatesuccinate (e.g., AQOAT (Shin Etsu)). The performance can vary based onthe degree and type of substitution. For example, HPMCP such as, HP-50,HP-55, HP-55S, HP-55F grades are suitable. The performance can varybased on the degree and type of substitution. For example, suitablegrades of hydroxypropylmethylcellulose acetate succinate include, butare not limited to, AS-LG (LF), which dissolves at pH 5, AS-MG (MF),which dissolves at pH 5.5, and AS-HG (HF), which dissolves at higher pH.These polymers are offered as granules, or as fine powders for aqueousdispersions; Poly Vinyl Acetate Phthalate (PVAP). PVAP dissolves inpH>5, and it is much less permeable to water vapor and gastric fluids.

In some embodiments, the coating may contain a plasticizer and possiblyother coating excipients such as colorants, talc, and/or magnesiumstearate, which are well known in the art. Suitable plasticizers includetriethyl citrate (Citroflex® 2), triacetin (glyceryl triacetate), acetyltriethyl citrate (Citroflec® A2), Carbowax® 400 (polyethylene glycol400), diethyl phthalate, tributyl citrate, acetylated monoglycerides,glycerol, fatty acid esters, propylene glycol, and dibutyl phthalate. Inparticular, anionic carboxylic acrylic polymers usually will contain10-25% by weight of a plasticizer, especially dibutyl phthalate,polyethylene glycol, triethyl citrate and triacetin. Conventionalcoating techniques such as spray or pan coating are employed to applycoatings. The coating thickness must be sufficient to ensure that theoral dosage form remains intact until the desired site of topicaldelivery in the intestinal tract is reached.

Colorants, detackifiers, surfactants, antifoaming agents, lubricants(e.g., carnuba wax or PEG) may be added to the coatings besidesplasticizers to solubilize or disperse the coating material, and toimprove coating performance and the coated product.

In some embodiments, a proliposomal powder dispersion disclosed hereinimproves the bioavailabity of testosterone. In various embodiments, afasting pharmacokinetic profile of mean plasma concentration oftestosterone ranges from 300 ng/dL to 1050 ng/dL (including, 400 ng/dLto 950 ng/dL, 500 ng/dL to 950 ng/dL, and 600 ng/dL to 950 ng/dL) oftestosterone five hours after administration under fasting conditions,i.e., ingestion of an oral dosage form described herein comprising 60 mgto 240 mg of testosterone. In some embodiments, a fastingpharmacokinetic profile of mean plasma concentration of testosterone isfrom about 300 ng/dL to about 1050 ng/dL of testosterone five hoursafter administration under fasting conditions, i.e., ingestion of anoral dosage form described herein comprising 60 mg to 240 mg oftestosterone. In some embodiments, a fasting pharmacokinetic profile ofmean plasma concentration of testosterone ranges from about 300 ng/dL toabout 1050 ng/dL (including, about 400 ng/dL to about 950 ng/dL, about500 ng/dL to about 950 ng/dL, and about 600 ng/dL to about 950 ng/dL) oftestosterone five hours after administration under fasting conditions,i.e., ingestion of an oral dosage form described herein comprising fromabout 60 mg to about 240 mg of testosterone. In some embodiments, afasting pharmacokinetic profile of mean plasma concentration oftestosterone is about 350 ng/dL of testosterone five hours afteradministration under fasting conditions, i.e., ingestion of an oraldosage form described herein comprising about 100 mg to about 260 mg oftestosterone. Such results represent as much as a 130 to 150 foldimprovement in the mean plasma concentration of testosterone as comparedto administration of equal dosages of unformulated testosterone. Adosage form described herein improves the bioavailibility oftestosterone under non-fasting, i.e., “fed” conditions. When a dosageform described herein is administered under fed conditions, the maximumplasma concentration (C_(max)) is about one half as much as the C_(max)under fasting conditions five hours after administration. In someembodiments, the C_(max) of the testosterone metabolite,dihydrotestosterone (DHT) is about 70 ng/dL after five hours. In someembodiments, the C_(max) of the testosterone metabolite,dihydrotestosterone (DHT) is 70 ng/dL after five hours for a 120 mgdose. In some embodiments, after about 24 hours under fed conditions theplasma concentration of testosterone following the administration of adosage form disclosed herein is greater than about 350 ng/dL, and thetestosterone metabolite, DHT, has a C_(max) of about 40 ng/dL, both ofwhich are above a typical normal ranges of endogenous testosterone andDHT, respectively, in a human. In some embodiments, after 24 hours underfed conditions the plasma concentration of testosterone following theadministration of a dosage form disclosed herein is 350 ng/dL, and thetestosterone metabolite, DHT5 has a C_(max) of 40 ng/dL, both of whichare above a typical normal ranges of endogenous testosterone and DHT,respectively, in a human.

Dose Amounts

In certain embodiments, the amount of testosterone in the pharmaceuticalcompositions is about 5 mg to about 1.0 g per dose, 10 mg to about 1.0 gper dose, about 50 mg to about 500 mg per dose. In some embodiments, theamount of testosterone in the pharmaceutical compositions is about 5 mgper dose, 10 mg per dose, about 50 mg per dose, about 100 mg per dose,about 120 mg per dose, about 150 mg per dose, about 180 mg per dose,about 210 mg per dose, about 240 mg per dose, about 270 mg per dose,about 300 mg per dose, about 350 mg per dose, about 400 mg per dose,about 450 mg per dose, about 500 mg per dose, or about 1000 mg per dose.In some embodiments, the amount of testosterone in the pharmaceuticalcompositions is about 120 mg per dose. In some other embodiments, theamount of testosterone in the pharmaceutical compositions is about 240mg per dose

In general, doses employed for adult human treatment are typically inthe range of 50 mg-1000 mg per day. In one aspect, doses employed foradult human treatment are from about 100 mg to about 300 mg per day. Insome embodiments, doses employed for adult human treatment are about 120mg per day. In some embodiments, doses employed for adult humantreatment are about 240 mg per day. In one embodiment, the desired doseis conveniently presented in a single dose or in divided dosesadministered simultaneously (or over a short period of time) or atappropriate intervals, for example as two, three, four or more sub-dosesper day.

In one embodiment, the daily dosages appropriate for testosterone isfrom about 0.01 to about 10 mg/kg per body weight. In other embodiments,the daily dosage or the amount of active in the dosage form are lower orhigher than the ranges indicated herein.

Methods of Dosing and Treatment Regimens

In some embodiments, the proliposomal powder dispersions and thepharmaceutical compositions disclosed herein are administered to anindividual in need of testosterone replacement therapy as often astestosterone replacement therapy is needed. In one embodiment, thepharmaceutical compositions described herein are administered forprophylactic and/or therapeutic treatments. In therapeutic applications,the compositions are administered to an individual already sufferingfrom a disease or condition, in an amount sufficient to remove allsymptoms or at least partially arrest at least one of the symptoms ofthe disease or condition. In certain embodiments, amounts effective forthis use depend on the severity and course of the disease or condition,previous therapy, the individual's health status, weight, and responseto the drugs, and/or the judgment of the treating physician.

In prophylactic applications, compositions described herein areadministered to an individual susceptible to or otherwise at risk of aparticular disease, disorder or condition. Such an amount is defined tobe a “prophylactically effective amount or dose.” In this use, theprecise amounts also depend on the individual's state of health, weight,and the like. When used in an individual, effective amounts for this usewill depend on the severity and course of the disease, disorder orcondition, previous therapy, the patient's health status and response tothe drugs, and the judgment of the treating physician.

In certain embodiments, administration of compositions or therapies asdescribed herein includes chronic administration. In certainembodiments, chronic administration includes administration for anextended period of time, including, e.g., throughout the duration of theindividual's life in order to ameliorate or otherwise control or limitthe symptoms of the individual's disease or condition. In someembodiments, chronic administration includes daily administration.

In some embodiments, administration of the compositions or therapiesdescribed herein is given continuously. In alternative embodiments, thedose of drug being administered is temporarily reduced or temporarilysuspended for a certain length of time (i.e., a “drug holiday”). In someembodiments, the length of the drug holiday varies between 2 days and 1year, including by way of example only, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250days, 280 days, 300 days, 320 days, 350 days, and 365 days. The dosereduction during a drug holiday is from 10%-100%, including by way ofexample only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, and 100%.

Once improvement of the individual's conditions has occurred, amaintenance dose is administered if necessary. Subsequently, in specificembodiments, the dosage or the frequency of administration, or both, isreduced, as a function of the symptoms, to a level at which the improveddisease, disorder or condition is retained. In certain embodiments,however, the individual requires intermittent treatment on a long-termbasis upon any recurrence of symptoms.

The amount of testosterone that corresponds to such an amount variesdepending upon factors such as the particular testosterone derivative,disease or condition and its severity, the identity (e.g., weight, sex)of the subject or host in need of treatment, but can nevertheless bedetermined according to the particular circumstances surrounding thecase, including, e.g., the specific agent being administered, the routeof administration, the condition being treated, and the subject or hostbeing treated.

Combination Treatments

In certain instances, it is appropriate to administer the proliposomalpowder dispersions and the pharmaceutical compositions disclosed hereinwith another therapeutic agent.

In general, the compositions described herein and, in embodiments wherecombinational therapy is employed, other agents do not have to beadministered in the same pharmaceutical composition, and are, because ofdifferent physical and chemical characteristics, administered bydifferent routes. In one embodiment, the initial administration is madeaccording to established protocols, and then, based upon the observedeffects, the dosage, modes of administration and times ofadministration, further modified.

In various embodiments, the multiple therapeutic agents are administeredconcurrently (e.g., simultaneously, essentially simultaneously or withinthe same treatment protocol) or sequentially, depending upon the natureof the disease, the condition of the patient, and the actual choice ofcompounds used. In certain embodiments, the determination of the orderof administration, and the number of repetitions of administration ofeach therapeutic agent during a treatment protocol, is based uponevaluation of the disease being treated and the condition of theindividual.

For combination therapies described herein, dosages of theco-administered therapeutic agents vary depending on the type of co-drugemployed, on the specific drug employed, on the disease or conditionbeing treated and so forth.

The individual therapeutic agents of such combinations are administeredeither sequentially or simultaneously in separate or combinedpharmaceutical formulations. In one embodiment, the individualtherapeutic agents will be administered simultaneously in a combinedpharmaceutical formulation. Appropriate doses of known therapeuticagents will be appreciated by those skilled in the art.

The combinations referred to herein are conveniently presented for usein the form of a pharmaceutical compositions together with apharmaceutically acceptable diluent(s) or carrier(s).

In some embodiments, the proliposomal powder dispersions and thepharmaceutical compositions disclosed herein are administered incombination with other therapeutic agents that reduce the severity of oreliminite the adverse effects associated with testosteronesupplementation. In some embodiments, adverse effects of testosteronesupplementation include acne and oily skin, increased hematocrit,exacerbation of sleep apnea and acceleration of pre-existing prostatecancer growth in individuals who have undergone androgen deprivation.Another adverse effect may be significant hair loss and/or thinning ofthe hair. Exogenous testosterone also causes suppression ofspermatogenesis and can lead to infertility.

In some embodiments, the proliposomal powder dispersions and thepharmaceutical compositions disclosed herein are administered incombination with other therapeutic agents that modulate testosteronemetabolism. In some embodiments, the other therapeutic agents reducetestosterone metabolism to dihydrotestosterone (DHT). In someembodiments, the other therapeutic agents reduce testosterone metabolismto estrogens (e.g. estradiol).

In some embodiments, the proliposomal powder dispersions and thepharmaceutical compositions disclosed herein are administered incombination with a synthetic 5-alpha-reductase inhibitor.5-Alpha-reductase inhibitors locks DHT, a byproduct of testosterone inthe body. 5-Alpha-reductase inhibitors include, but are not limited to,finasteride, alfatradiol, and dutasteride.

In some embodiments, the proliposomal powder dispersions and thepharmaceutical compositions disclosed herein are administered incombination with clomifene.

Gonadotropin and testosterone therapy is available in treatment ofhypogonadism in men. The treatment strategy depends on the age ofpatient and the goals of therapy (restore of fertility and/or produceand maintain of virilization). The gonadototropins and GnRH are usefulin spermetogenesis stimulation. In some embodiments, the proliposomalpowder dispersions and the pharmaceutical compositions disclosed hereinare administered in combination with gonadotropins and/or GnRH.

In some embodiments, the compositions and methods described herein arealso used in conjunction with estrogen inhibitors, for example aromataseinhibitors. In some embodiments, the proliposomal powder dispersions andthe pharmaceutical compositions disclosed herein are administered incombination with an aromatase inhibitor or combinations of aromataseinhibitors. Exemplary aromatase inhibitors include, but are not limitedto, aminoglutethimide, testolactone, anastrozole, letrozole, exemestane,vorozole, formestan, fadrozole, 4-hydroxyandrostenedione,1,4,6-Androstatrien-3,17-dione (ATD), and 4-Androstene-3,6,17-trione(“6-OXO”).

In one embodiment, the compositions and methods described herein arealso used in conjunction with other therapeutic reagents that areselected for their particular usefulness against the condition that isbeing treated. For example, the compositions disclosed herein may beadministered in combination with insulin where the conditions istestosterone deficiency associated with diabetes; the composition may beadministered in combination with calcium or an osteoporosis medicationwhere the condition to be treated is testosterone deficiency associatedwith osteoporisis; the compositions may be administered in combinationwith an HIV/AIDS medication where the condition to be treated istestosterone deficiency associated with HIV/AIDS; the compositions maybe administered in combination with an chemotherapy or radiation therapywhere the condition to be treated is testosterone deficiency associatedwith cancer.

Exemplary osteoporosis medications include calcium, calcitonin,parathyroid hormone, recombinant parathyroid hormone (e.g.,teriparatide), a RANKL inhibitor (e.g., denosumab), a bisphosphonate(e.g., etidronate, clodronate, tiludronate, pamidronate, neridronate,olpadronate, alendronate, ibandronate, risedronate, zoledronate).

Exemplary HIV/AIDS medications include abacavir; amprenavir; atazanavir;darunavir; delavirdine; didanosine; edurant; efavirenz; emtricitabine;enfuvirtide; etravirine; fosamprenavir; indinavir; lamivudine;lopinavir; maraviroc; nelfinavir; nevirapine; raltegravir; ritonavir;saquinavir; stavudine; tenofovir disoproxil fumarate (DF); tipranavir;zalcitabine; zidovudine.

Exemplary chemotherapeutics include nitrogen mustards such as forexample, bendamustine, chlorambucil, chlormethine, cyclophosphamide,ifosfamide, melphalan, prednimustine, trofosfamide; Alkyl Sulfonateslike busulfan, mannosulfan, treosulfan; Ethylene Imines like carboquone,thiotepa, triaziquone; Nitrosoureas like carmustine, fotemustine,lomustine, nimustine, ranimustine, semustine, streptozocin; Epoxidessuch as for example, etoglucid; Other Alkylating Agents such as forexample dacarbazine, mitobronitol, pipobroman, temozolomide; Folic AcidAnalogues such as for example methotrexate, permetrexed, pralatrexate,raltitrexed; Purine Analogs such as for example cladribine, clofarabine,fludarabine, mercaptopurine, nelarabine, tioguanine; Pyrimidine Analogssuch as for example azacitidine, capecitabine, carmofur, cytarabine,decitabine, fluorouracil, gemcitabine, tegafur; Vinca Alkaloids such asfor example vinblastine, vincristine, vindesine, vinflunine,vinorelbine; Podophyllotoxin Derivatives such as for example etoposide,teniposide; Colchicine derivatives such as for example demecolcine;Taxanes such as for example docetaxel, paclitaxel, paclitaxelpoliglumex; Other Plant Alkaloids and Natural Products such as forexample trabectedin; Actinomycines such as for example dactinomycin;Antracyclines such as for example aclarubicin, daunorubicin,doxorubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin,valrubicin, zorubincin; Other Cytotoxic Antibiotics such as for examplebleomycin, ixabepilone, mitomycin, plicamycin; Platinum Compounds suchas for example carboplatin, cisplatin, oxaliplatin, satraplatin;Methylhydrazines such as for example procarbazine; Sensitizers such asfor example aminolevulinic acid, efaproxiral, methyl aminolevulinate,porfimer sodium, temoporfin; Protein Kinase Inhibitors such as forexample dasatinib, erlotinib, everolimus, gefitinib, imatinib,lapatinib, nilotinib, pazonanib, sorafenib, sunitinib, temsirolimus;Other Antineoplastic Agents such as for example alitretinoin,altretamine, amzacrine, anagrelide, arsenic trioxide, asparaginase,bexarotene, bortezomib, celecoxib, denileukin diftitox, estramustine,hydroxycarbamide, irinotecan, lonidamine, masoprocol, miltefosein,mitoguazone, mitotane, oblimersen, pegaspargase, pentostatin,romidepsin, sitimagene ceradenovec, tiazofurine, topotecan, tretinoin,vorinostat; Estrogens such as for example diethylstilbenol,ethinylestradiol, fosfestrol, polyestradiol phosphate; Progestogens suchas for example gestonorone, medroxyprogesterone, megestrol; GonadotropinReleasing Hormone Analogs such as for example buserelin, goserelin,leuprorelin, triptorelin; Anti-Estrogens such as for examplefulvestrant, tamoxifen, toremifene; Anti-Androgens such as for examplebicalutamide, flutamide, nilutamide, Enzyme Inhibitors,aminoglutethimide, anastrozole, exemestane, formestane, letrozole,vorozole; Other Hormone Antagonists such as for example abarelix,degarelix; Immunostimulants such as for example histaminedihydrochloride, mifamurtide, pidotimod, plerixafor, roquinimex,thymopentin; Immunosuppressants such as for example everolimus,gusperimus, leflunomide, mycophenolic acid, sirolimus; CalcineurinInhibitors such as for example ciclosporin, tacrolimus; OtherImmunosuppressants such as for example azathioprine, lenalidomide,methotrexate, thalidomide; and Radiopharmaceuticals such as for example,iobenguane; immunostimulants such as for example ancestim, filgrastim,lenograstim, molgramostim, pegfilgrastim, sargramostim; interferons suchas for example interferon alfa natural, interferon alfa-2a, interferonalfa-2b, interferon alfacon-1, interferon alfa-n1, interferon betanatural, interferon beta-1a, interferon beta-1b, interferon gamma,peginterferon alfa-2a, peginterferon alfa-2b; Interleukins such as forexample aldesleukin, oprelvekin; Other immunostimulants such as forexample BCG vaccine, glatiramer acetate, histamine dihydrochloride,immunocyanin, lentinan, melanoma vaccine, mifamurtide, pegademase,pidotimod, plerixafor, poly I:C, poly ICLC, roquinimex, tasonermin,thymopentin; Immunosuppressants such as for example abatacept, abetimus,alefacept, antilymphocyte immunoglobulin (horse), antithymocyteimmunoglobulin (rabbit), eculizumab, efalizumab, everolimus, gusperimus,leflunomide, muromab-CD3, mycophenolic acid, natalizumab, sirolimus; TNFalpha Inhibitors such as for example adalimumab, afelimomab,certolizumab pegol, etanercept, golimumab, infliximab; InterleukinInhibitors such as for example anakinra, basiliximab, canakinumab,daclizumab, mepolizumab, rilonacept, tocilizumab, ustekinumab;Calcineurin Inhibitors such as for example ciclosporin, tacrolimus;immunosuppressants such as for example azathioprine, lenalidomide,methotrexate, thalidomide; adalimumab, Alemtuzumab, Basiliximab,Bevacizumab, Cetuximab, Certolizumab pegol, Daclizumab, Eculizumab,Efalizumab, Gemtuzumab, Ibritumomab tiuxetan, Infliximab, Muromonab-CD3,Natalizumab, Panitumumab, Ranibizumab, Rituximab, Tositumomab,Trastuzumab, or the like, or a combination thereof.

Kits/Articles of Manufacture

For use in the therapeutic methods of use described herein,kits/articles of manufacture are also described herein. Such kitsinclude a carrier, package, or container that is optionallycompartmentalized to receive one or more doses of a pharmaceuticalcomposition of testosterone as described herein. The kits providedherein contain packaging materials. Packaging materials for use inpackaging pharmaceutical products include, but are not limited to thosedescribed in e.g., U.S. Pat. No. 5,323,907. Examples of pharmaceuticalpackaging materials include, but are not limited to, blister packs,bottles, tubes, bags, containers, bottles, and any packaging materialsuitable for a selected formulation and intended mode of administrationand treatment. A wide array of formulations of the compounds andcompositions provided herein are contemplated as are a variety oftreatments for any disease, disorder, or condition that would benefit bytreatment with testosterone replacement therapy.

For example, the container(s) include the proliposomal powderdispersions and the pharmaceutical compositions disclosed herein aloneor in combination with another agent as disclosed herein. Such kitsoptionally include an identifying description or label or instructionsrelating to its use in the methods described herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. The pack, for example, containsmetal or plastic foil, such as a blister pack. In one embodiment, thepack or dispenser device is accompanied by instructions foradministration. In one embodiment, the pack or dispenser is alsoaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, is the labeling approved by the U.S. Food andDrug Administration for prescription drugs, or the approved productinsert. In one embodiment, compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

EXAMPLES

The following ingredients, formulations, processes and procedures forpracticing the methods disclosed herein correspond to that describedabove. The procedures below describe with particularity illustrative,non-limiting embodiments of formulations that include testosterone andmethods of uses thereof.

Comparative Example 1 Preparation of Testosterone DMPG ProliposomalFormulations

Comparative Proliposomal Formulations (“PLF-C”), PLF-C1, PLF-C2, PLF-C3,and PLF-C4 contained testosterone and dimyristyl phosphatidyl glycerolsodium (DMPG). Table 1 lists the components and amounts of each used tomake each of these formulations. For PLF-C1, 0.5 g of testosterone wasdissolved in 3 ml of a 9:1 ethanol to water mixture (v/v), and 0.5 g ofDMPG was added to this solution. The solution was then stirred at roomtemperature until complete evaporation of the solvent was achieved,typically overnight. The powder formulation obtained upon evaporation ofthe solvent was passed through an appropriate sieve, such as mesh #60.The sieved powder was then transferred to a glass vial and stored atroom temperature while protected from light.

The preparations of PLF-C2 and PLF-C3 were like that of PLF-C1, exceptthat for PLF-C2, 1.5 g of testosterone and 1.5 g of DMPG were dissolvedin 20 ml of the 9:1 ethanol-water solution, and for PLF-C3, 0.5 gtestosterone and 1 g DMPG were dissolved in 10 ml of the 9:1ethanol-water solution.

Formulation PLF-C4 was also prepared in the same way as PLF-C1 wasprepared, except that 0.5 g of testosterone, and 0.5 g of DMPG weredissolved in 12 ml of an 1:1:0.3 mixture (v/v) of chloroform, ethanol,and water, respectively.

The above formulations were each filled into size “00” hard gelatincapsules, and then tested for their dissolution. Only PLF-C1 capsuleswere enteric coated. Specifically, the PLF-C1 coating material wasEudragit® L 30 D-55. Coating of PLF-C1 capsules was performed by firstpreparing a dispersion of Eudragit® L 30 D-55. To this dispersion wasadded amount of triethyl citrate equivalent to 10% of Eudragit® polymerweight to enhance film formation. Each capsule was dipped into thedispersion and then air dried a total of four times. Some of thecapsules from formulation PLF-C1 were not coated. These uncoatedcapsules were also used in dissolution testing along with enteric coatedcapsules.

TABLE 1 PLF-C1 PLF-C2 PLF-C3 PLF-C4 Ingredients Qty Qty Qty QtyTestosterone 0.50 g 1.50 g 0.50 g 0.50 g DMPG 0.50 g 1.50 g 1.00 g 0.50g Ethanol:Water (9:1) 3 ml 20 ml 10 ml — mixture Chloroform-ethanol- — —— 12 ml water mixture (1:1:0.3) Drug:Lipid Ratio 1:1 1:1 1:2 1:1

Comparative Example 2 In Vitro Dissolution of Testosterone DMPGProliposomal Formulations

In vitro dissolution profiles of testosterone from enteric-coated anduncoated PLF-C1 capsules were obtained. For these studies, threedifferent dissolution media were used for enteric coated capsules. Theenteric coated capsules (n=3) were dissolved in 500 ml of either: 1) 0.1N HCl (pH 1.20) for 1 h; sodium acetate buffer (pH 4.50) for 1 h;potassium phosphate buffer (pH 6.80) for 3 h. For uncoated capsules,(n=3), only 0.1N HCl (pH 1.20) was used and samples were collected after1, 2 and 2.5 h. Dissolution was allowed to proceed in a USP type-1apparatus at a speed of 50 rotations per minute (rpm) at 37° C. for 0.5h, 1 h, 2 h, and 3 h. At each timepoint, 5 ml samples were collected.The amounts of testosterone that were released at each time point weredetermined by using the following high pressure liquid chromatography(HPLC) assay method to analyze each timepoint sample. For eachformulation analyzed, an amount of the formulation that contains 25 mgof testosterone was dissolved in 25 ml of ethanol. This stock solutionwas further diluted suitably with a methanol: water (1:1) solution untilthe concentration of testosterone reached 10 μg/ml. A 20 μl aliquot wasthen injected into the HPLC. The composition of mobile phase used in theHPLC analysis was a solution of acetonitrile:(water with 0.2% formicacid) (75:25 v/v). Separation of the phases was achieved by using a C18(100×4.6 mm; 5 μm) Kinetex, Phenomenex® column at a flow rate of 1.0ml/min. The total run time for each sample was 5 min and the Photo DiodeArray detector (PDA) detector was set at a wavelength of 243 nm. Bothuncoated and coated capsule formulations showed a drug release of lessthan 3.46% in 3 hours in PBS pH 6.80. Enteric-coated capsules remainedintact for 2 hours in acidic pH.

In vitro dissolution profiles of testosterone from uncoated PLF-C2capsules were also obtained. For these studies, uncoated capsules weredissolved in 900 ml of potassium phosphate buffer (pH 6.80). Thedissolution and analysis parameters that were used were the same asdescribed above for the PLF-C1 studies. The in vitro release profiles oftestosterone from PLF-C2 uncoated capsules stored in screw-capped glassvials at different temperatures is shown in Table 2 and FIG. 1. Thedifferences between the testosterone release profiles of PLF-C2 andPLF-C4 uncoated capsules, which both comprise DMPG in different ratios,are shown in FIG. 2.

TABLE 2 Time of Storage storage % Drug dissolved at each time point(hours) Formulation Temp. (months) % Assay 0.5 1 2 3 PLF-C2 0, Initial138.23 0.46 ± 0.64 1.15 ± 0.70 1.88 ± 0.91 2.38 ± 1.03 (D:DMPG is RT 1123.81 — — — — 1:1) 30° C. 1 118.02 — — — — RT 2 — — — — — 30° C. 2122.92 — — — — RT 3 122.80 0.30 ± 0.03 0.76 ± 0.07 1.39 ± 0.26 1.88 ±0.48 30° C. 3 137.72 0.38 ± 0.13 1.00 ± 0.23 1.98 ± 0.38 2.53 ± 0.43

Comparative Example 3 In Vivo Testing of Testosterone DMPG ProliposomalFormulations

Formulations PLF-C1, PLF-C2, and PLF-C3 were tested for their in vivoperformance following oral administration in rats (n=3). These studiesused an institutional animal ethical committee (IACUC)-approved in vivoprotocol involving male Sprague Dawley® rats (Charles Rivers—Wilmington,Mass.) weighing approximately 250 grams each were canulated in thejugular vein and were used for the study. The PLF-C1 formulation in itsfree-flowing proliposomal powder form, and a testosterone controlsolution (a 0.5% (wt/vol) dispersion of testosterone in an HPMCsuspension were tested in fed rats after oral administration.

The PLF-C1 and testosterone control solutions were prepared bydispersing them in a 0.5% (w/v) hydroxypropylmethyl cellulose (HPMC)suspension. Fed rats were administered 1 ml of pure drug solution (300mg/Kg body weight dose) and 2.5 ml of PLF-C1 formulation (300 mg/Kg bodyweight dose) by oral gavage, respectively. Blood samples were collectedat 0, 1, 2, 4, 8, 12, 24 hours of dosing. The samples were stored at 4°C. until centrifuged. Plasma portions of the blood samples wereseparated from blood by centrifugation at 12,000 rpm and 4° C. for 15min using a Microfuge® 22R centrifuge (Beckman-Coulter). Plasma sampleswere stored at −20° C. until they were analyzed.

In vivo studies were also performed using the PLF-C2 and PLF-C3formulations. These formulations were administered orally to male SDrats fasted overnight with free access to water. The animals weredivided into two groups each comprising of 3 animals each. FormulationPLF-C2 was administered to one group while PLF-C3 was given to the othergroup.

To prepare PLF-C2 and PLF-C3 for administration, amounts of eachsufficient to administer the experimental dose of 300 mg/kg of bodyweight to five rats was weighed and dispersed in 20 ml of water. Ratswere administered 4 ml of the respective suspended formulations. Bloodsamples were collected at time 0 (before dosing), as well as at 1, 2, 4,8, 12 and 24 hours after administration. The collected samples werestored in an ice-box until centrifuged at 12,000 rpm for 15 min. at 4°C. Plasma was separated and stored at −20° C. until it was analyzed.Detection of testosterone in the plasma samples was performed by using avalidated liquid chromatography tandem mass spectrometry method(LC-MS/MS) as described below.

HPLC-eluted samples were prepared for MS analysis of testosterone andDHT concentrations. Briefly, the HPLC system used was a ShimadzuCLASS-VP® System. The mobile phase solutions were (A) 0.2% formic acidin water, and (B) 0.2% formic acid in acetonitrile. The column used inthe analysis was a 2×10 mm Duragel® G C18 guard cartridge (PeekeScientific—Novato, Calif.). The injection volume was 25 μl, the gradientwas determined by going from a 5% to a 30% solution of buffer (B) in 0.5minutes, followed by going from a 30% to a 55% solution of (B) in 2minutes. The flow rate was 400 μl/min.

Internal standards, i.e., calibration standards, quality control (QC)samples and HPLC-eluted plasma samples were prepared for LC/MS/MSanalysis by precipitating 50 μl of each sample with 2× volumes of icecold Internal Standard Solution (acetonitrile containing 50 ng/ml oftestosterone 16, 16, 17-d₃, d₃-testosterone). In addition tod₃-testosterone serving as an internal standard, dihydrotestosterone(DHT) was used as a QC standard. The precipitated samples werecentrifuged at 6100×g for 30 minutes (or equivalent). Followingcentrifugation, 100 μl of each supernatant was transferred to anauto-sampler plate and placed on a heating block for 1 hour to partiallyevaporate the acetonitrile in the samples. MS analysis of the sampleswas then performed according to the following parameters.

The testosterone detection step of the analysis was performed by usingan Applied Biosystems/MDS SCIEX API 3000®-equipped with a TurbolonSpray®(Applied Biosystems) electro spray interface (ESI) system The flow ofliquid solvent from the analytical column entered the heated nebulizerinterface of the MS/MS analyzer. The above-solvent/sample mixture wasfirst converted to vapor in the heated tubing of the interface at 400°C. The analytes (testosterone, DHT, and [D3]-testosterone), contained inthe nebulized solvent, were ionized and a positive charge added by thecorona discharge needle of the interface, which applies a large voltageto the nebulized solvent/analyte mixture. The ions passed through theorifice of the instrument and entered the first quadrapole. Quadrapoles1 and 3 (Q1 and Q3) were the mass filters, allowing selection of ionsbased on their mass to charge ratio (m/z). Quadrapole 2 (Q2) was thecollision cell, where ions were fragmented by collision with argonmolecules.

The first quadrapole of the MS/MS (Q1) selected for testosterone with anm/z value of 289.2, DHT with an m/z value of 291.2, or the internalstandard, d₃-testosterone, with an m/z value of 292.2. Ions with thesem/z values passed to the collision chamber (Q2), while ions with anyother m/z collided with the sides of the quadrapole and were destroyed.Ions entering Q2 collided with neutral gas molecules. This process iscalled Collision-Induced Dissociation (CID). The CID gas used in thisexample was argon. The daughter ions generated were passed intoquadrapole 3 (Q3), where the daughter ions of testosterone with an m/zvalue of 96.9, DHT with an m/z value of 255.2, or those of testosterone16, 16, 17-d₃ (d₃-testosterone), the internal standard, with an m/zvalue of 96.9 were selected for, while other ions were screened out. Theselected daughter ions were collected by the detector. Quantification isbased on peak area ratio of analytes, i.e., testosterone, over theinternal standard acquired by selective reaction monitoring (SRM) inpositive mode. The mean plasma concentration over time profiles of thePLF-C1 formulation and testosterone control solution under fedconditions are shown in FIG. 3. The mean plasma concentration over timeprofiles of the PLF-C2 and PLF-C3 formulations under fasting conditionsare shown in FIG. 4.

Comparative Example 4 Dissolution of DMPG-Based Formulations with AvicelPH101 and Lactose Monohydrate

For formulation PLF-C5, testosterone was dissolved in a mixture ofethanol:water (9:1). To this solution dimyristyl phosphatidyl glycerolsodium (DMPG) and lactose monohydrate were added and stirred overnightuntil complete evaporation of the solvent at room temperature. Lactosewas added so that it helps in dispersion and avoids lump formation whilepreparing the dosing solution for animal studies.

Another formulation (PLF-C5+Avicel® PH101) was prepared by addingAvicel® PH101 externally to the formulation (PLF-C5) containing drug:DMPG: lactose (1:1:1). This approach was followed to see if Avicel®PH101 helps in increasing the dispersability of the formulation duringdissolution.

In yet another example, formulation (PLF-C5+Avicel® PH101+Explotab®disintegrant) was prepared by adding Avicel® and Explotab® disintegrant(1%) externally to PLF-C5. This formulation was prepared with theobjective of increasing the dispersion and there by the dissolution ofthe proliposomal formulation with the help of super disintegratingagent, Explotab® disintegrant and better dispersability of Avicel®PH101.

In yet another example, formulation PLF-C2 containing testosterone: DMPGin 1:1 ratio was taken and dispersed in 5 ml dissolution media first andthen dissolution was performed. This was done to compare the dissolutionof encapsulated PLF-C2 to dispersed PLF-C2 because, prior to animaldosing, the formulation was dispersed in water.

In yet another example, formulation PLF-C6 was prepared by dissolvingtestosterone in a mixture of ethanol: water (9:1). To the resultingtestosterone solution, DMPG and Avicel® PH 101 were added at a ratio of1:1:2, and dispersed by stirring the mixture overnight until thecomplete evaporation of the solvent had occured at room temperature toform a proliposomal powder. To the dried proliposomal formulation,Explotab® disintegrant was added externally and the powder-Explotab®disintegrant mixture was formulated as a capsule. The objective was tosee the effect of using dispersed Avicel® PH101 on dissolution ofproliposomal formulation.

A testosterone proliposomal formulation was lyophilized. Specifically,formulation PLF-C5 (500 mg) containing testosterone: DMPG: lactose(1:1:1) was taken and dispersed in 20 ml of water. It was hydrated at40° C. for 30 min, and then lyophilized for more than 24 hours. Thecompositions of the variations of PLF-C5 that were created by addingvarious excipients used are listed in Table 3.

In vitro dissolution studies of PLF-C5 and PLF-C5 formulationscomprising additional excipients was carried out by using apparatus typeI at 50 rpm. The dissolution media used was 900 ml of phosphate buffer(pH 6.80) that was maintained at 37±0.5° C. Samples (5 ml) werecollected after 1, 2, 3 and 4 hours of dissolution. A type II apparatuswas used for the dissolution of all other formulations in Table 3. Allother dissolution parameters were same as that of PLF-C5, describedabove.

Testosterone dissolution assays of the foregoing PLF-C5 formulationswere tested as per the method described in Comparative Example 2. The invitro release profile of all the DMPG formulations described inComparative Example 4 are shown in FIG. 5. As shown in FIG. 5,formulations containing Avicel® and Explotab® disintegrant gave higherrelease as compared to others.

TABLE 3 (d) (a) PLF-C2 (e) (f) Ingredients PLF-C5 (b) (c) hydratedPLF-C6 Lyophilized Testosterone 1.0 g 0.10 g 300 mg 50 mg 1.0 g 0.50 gDMPG (NOF Corp. 1.0 g (PLF-C5) 50 mg 1.0 g Japan) Lactose monohydrate1.0 g — — Avicel PH 101 — 0.10 g 600 mg — 1.0 g — Explotab —  9 mg —0.01 g  — Ethanol:Water (9:1)   25 ml   20 ml — — — — mixtureDissolution media — — —  5 ml — Water — — — — —   20 ml

Example 1 Solubility Studies in Various Buffers

The solubility of testosterone in different media was tested. An amountof testosterone (100 mg) was added to each of four glass vialscontaining 10 ml of either: a) HCl buffer (pH 1.20), b) Acetate buffer(pH 4.50), c) phosphate buffer solution (PBS) pH 6.80 and d) nano purewater. These samples were subjected for shaking in a water bath for 72 hat RT. The samples were suitably diluted and analyzed by the HPLC assaymethod described in Comparative Example 2. Solubility of testosteronewas high in phosphate buffer pH 6.80. See FIG. 6.

The solubility of testosterone was measured in the presence of lipidcomponents at 37° C. in aqueous medium (pH 6.8) under the in vitrodissolution conditions described in Comparative Example 2. Physical mixformulations were prepared by mixng the dry components as per thecompositions given in Table 4 (F1-F9). Dissolution parameters used forthese studies are listed below. Samples collected at each time pointwere diluted suitably and analyzed by the HPLC assay method given inComparative Example 2.

The dissolution parameters for these dissolution studies were asfollows. Formulations were dissolved using an USP Type 2 (Paddle)apparatus in a volume of 1000 ml of potassium phosphate buffer pH 6.80(USP 30). Dissolution occurred at 37° C., and at 75 rpm. Samples (5 ml)were taken for analysis at 3, 5, 8 10, 12, 18 and 24 hours. Thedissolution curve is shown in FIG. 7. Formulations F7 and F9 withmaximum amount of DSPC in the range of 48% to 60% (w/w) showed highsolubility in PBS (pH 6.80). See FIG. 7.

TABLE 4 Testosterone DSPC Cholesterol Formulation Wt % mg/cap Wt %mg/cap Wt % mg/cap F1 60 144.00 32 76.8 8 19.20 F2 60 144.00 36 86.4 49.60 F3 60 144.00 40 96.0 0 0 F4 50 120.0 40 96.0 10 24.0 F5 50 120.0045 108.0 5 12.00 F6 50 120.00 50 120.0 0 0 F7 40 96.00 48 115.20 1228.80 F8 40 96.00 54 129.60 9 21.60 F9 40 96.00 60 144.00 0 0

Example 2 Preparation of Testosterone/Cholesterol DSPC ProliposomalFormulations

Proliposomal Formulation (PLF)-1, PLF-2, PLF-3, and PLF-4 were preparedby dissolving testosterone in a 9:1 mixture (v/v) of ethanol and water,followed by the addition of distearoyl phosphatidylcholine (DSPC) andcholesterol at a 9:1 ratio of DSPC to cholesterol (w/w). The resultingdispersion was stirred overnight until complete evaporation of thesolvent at room temperature. The composition of these formulations isdetailed in Table 5. The powder obtained upon removal of the solvent waspassed through an appropriate sieve (mesh #60), transferred to a glassbottle, and stored at room temperature protected from light.

TABLE 5 Ingredients PLF-1 PLF-2 PLF-3 PLF-4 Testosterone 1.00 g 1.50 g1.50 g 1.0 g DSPC 0.90 g 1.35 g 2.70 g 1.350 g Cholesterol 0.10 g 0.150g 0.30 g 0.150 g Ethanol:Water (9:1) 20 ml 20 ml 20 ml 20 ml mixtureDrug:Lipid ratio 1:1 1:1 1:2 1:1.5 Lipid (DSPC + Cholesterol)

Example 3 Solubility Studies

Solubility of: a) 10 mg pure testosterone; and b) formulation PLF-2equivalent to 10 mg testosterone in either 0.5%, 1%, or 2% SLS followingsonication for 10 min. was determined. For these studies, 10 mg of puretestosterone was weighed and dissolved in either 100 ml of 0.5%, 1%, or2% (w/v) SLS, respectively, in volumetric flasks. An amount offormulation PLF-2 that contained 10 mg of testosterone was weighed, andsample solutions were prepared in a similar way as that described abovefor the testosterone control. These solutions were subjected forsonication for 10 min in a bath sonicator. The samples were thenfiltered and diluted suitably in their respective concentrations of SLS.The amounts of soluble testosterone for each concentration of SLS wasdetermined by using the HPLC assay method described in ComparativeExample 2. Both the testosterone control and PLF-2 were associated withhigh testosterone solubility in 1% w/v SLS. See FIG. 8.

Example 4 In Vitro Dissolution Studies of Testosterone/Cholesterol DSPCProliposomal Formulations

Formulation PLF-1 was filled into size “00” hard gelatin capsules. Someof these capsules enterically coated as described in Comparative Example2. Dissolution of both enteric-coated (n=3) and uncoated capsules (n=3)was carried out in 900 ml of potassium phosphate buffer (pH 6.80), anddissolution conditions were maintained throughout a three hour timecourse at 37±0.5° C. Samples (5 ml) were collected after 0.5, 1, 2 and 3hours of dissolution, and then assayed according to the HPLC assaymethod as described above in Comparative Example 2. The stability dataof PLF-1 is summarized in Table 6 and the in vitro release profile isgiven in FIG. 9.

TABLE 6 Formulation Storage stored in Storage Time % Drug dissolved attime points glass vial Condition (Months) % Assay 0.5 h 1 h 2 h 3 hPLF-1 Initial 90.50 3.54 ± 1.54 7.15 ± 4.66 13.25 ± 9.59  18.01 ± 12.34D:DSPC:CH RT 1 94.03 — — — — (1:0.9:0.1) 30° C. 1 99.17 — — — — RT 2 — —— — — 30° C. 2 110.33 — — — — RT 3 91.40 3.86 ± 5.39 8.92 ± 9.65 18.27 ±8.29 24.58 ± 6.11 30° C. 3 101.06 5.97 ± 2.60 13.49 ± 2.24  23.41 ± 1.2929.17 ± 2.78

Example 5 In Vivo Testing of Testosterone/Cholesterol DSPC ProliposomalFormulations

The formulations PLF-2 and PLF-4 were prepared according to the methodof their preparation described in Example 2. The formulations were thenadministered orally to male Sprague Dawley® (SD) rats after an overnightfast. Formulation PLF-2 was tested at 300 mg/kg, 150 mg/kg, 31 mg/kg,15.5 mg/kg and 7.75 mg/kg doses of testosterone to body weight.Formulation PLF-4 was administered orally in a dose of 300 mg/kg bodyweight.

Male SD rats, which were canulated in the jugular vein and weighedapproximately 250 grams were used for the study. Animals were fastedovernight with free access to water. Sampling and analysis was followedas per the method described in paragraphs [063-067] under comparativeExample 4. The mean plasma concentration vs time profiles of theformulations that were tested are shown in FIGS. 10 and 11. The plasmaconcentrations of testosterone following the oral administration ofdifferent test formulations and a control testosterone dispersion werefollowed over time. Both 300 mg/Kg and 31 mg/Kg (testosterone dose/bodyweight) control and test formulations were studied.

Example 6 Dissolution of Testosterone/Cholesterol DSPC ProliposomalFormulations that Comprise Avicel® PH 101, Lactose Monohydrate,Magnesium Stearate

A base Formulation called PLF-5 was prepared by dissolving 1.5 mg oftestosterone in 20 ml of a 9:1 mixture (v/v) of ethanol and water,followed by the addition of 1.35 mg of distearoyl phosphatidylcholine(DSPC) and 0.15 mg of cholesterol, (i.e., a 9:1 ratio of DSPC tocholesterol (w/w)). The resulting dispersion was stirred overnight untilcomplete evaporation of the solvent at room temperature. The powderobtained upon removal of the solvent was passed through an appropriatesieve (mesh #60), transferred to a glass bottle, and stored at roomtemperature protected from light. The PLF-5 formulation was then furtherformulated into PLF-5 formulations (a-d), as shown in Table 7, anddescribed below.

PLF-5 (a) was prepared in capsule form, and was made with the objectiveof determining whether Avicel® PH101 and Explotab® disintegrant wouldincrease the dispersability of the formulation during dissolution. PLF-5(a) was prepared by the external addition (i.e., mixing the excipientsand blending them with the Testosterone/Cholesterol DSPC ProliposomalFormulations) of the following ingredients to PLF-5: Avicel® PH 101 at aratio of 1:2 (w/w); and Explotab® in the amount of 1% (w/w). Theformulation mixture was then filled into capsules.

PLF-5 (b) was prepared in tablet form, and was made with the objectiveof determining whether the addition of Avicel® PH101, Explotab®,magnesium (Mg) stearate, and spray-dried lactose monohydrate wouldincrease the dispersability of the formulation during dissolution. PLF-5(b) was prepared by the external addition of the following ingredientsto PLF-5: Avicel® PH 101 at a ratio of 1:2 (w/w); Explotab® in theamount of 1% (w/w); Spray-dried lactose monohydrate in an amountequivalent to 0.04 g per tablet; and Mg stearate in an amount equivalentto 8.75 mg per tablet. The mixture was formulated into an 8.9 mmcircular biconvex tablet weighing 350 mg.

PLF-5 (c), which contained twice the amount of PLF-5 per dose, thanPLF-5 (a) and (b) formulations, was prepared by the external addition ofthe following ingredients to PLF-5: Avicel® PH 101 at a ratio of 1.5:1(w/w); Explotab® in the amount of 5% (w/w); and Mg stearate in an amountequivalent to 0.00875 g per tablet. The mixture was formulated into an8.9 mm circular biconvex tablet weighing 350 mg.

PLF (d) was prepared exactly as PLF (c), but it was not tableted.

The in vitro testosterone dissolution profiles for the formulationsPLF-5 (a-d) are provided in FIG. 12. Formulations containing Avicel®along with Explotab® disintegrant showed better dissolution profiles. Inaddition, both 1 wt % and 5 wt % Explotab® disintegrant-containingformulations showed similar release profiles.

The profiles were determined by using a type II apparatus at 50 rpm in900 ml of PBS (pH 6.80) to dissolve the formulations. Samples werecollected after 1, 2, 3 and 4 hours of dissolution, and were analyzed bythe HPLC assay method described in Comparative Example 2.

TABLE 7 Ingredients PLF-5 (a) PLF-5 (b) PLF-5 (c) PLF-5 (d) PLF-5Capsule Tablet Tablet Powder 1.5 g Testosterone 100 mg 100 mg  200 mg 200 mg 1.35 g DSPC 0.15 g Cholesterol Lactose monohydrate —  45 mg — —spray dried (SD) Avicel ® PH 101 200 mg 200 mg  131 mg  131 mgExplotab ®  3 mg  3.5 mg 17.5 mg 17.5 mg Magnesium stearate — 8.75 mg 8.75 mg 8.75 mg

Example 7 Dissolution of Testosterone/Cholesterol DSPC ProliposomalFormulations that Comprise Avicel® PH 101/102, Lactose Monohydrate, MgStearate, and Pearlitol® 200 SD

A base Formulation called PLF-6 was prepared by dissolving 3 mg oftestosterone in 40 ml of a 9:1 mixture (v/v) of ethanol and water,followed by the addition of 2.7 mg DSPC and 0.30 mg of cholesterol,(i.e., a 9:1 ratio of DSPC to cholesterol (w/w)). The resultingdispersion was stirred overnight until complete evaporation of thesolvent at room temperature. The powder obtained upon removal of thesolvent was passed through an appropriate sieve (mesh #60).

A second, base formulation called PLF-7 was also prepared for thesestudies by dissolving 1.2 mg of testosterone in 30 ml of a 9:1 mixture(v/v) of ethanol and water, followed by the addition of 1.08 mg DSPC and0.12 mg of cholesterol, (i.e., a 9:1 ratio of DSPC to cholesterol(w/w)). The resulting dispersion was stirred overnight until completeevaporation of the solvent at room temperature. The powder obtained uponremoval of the solvent was passed through an appropriate sieve (mesh#60).

PLF-6 and PLF-7 were further formulated into PLF-6 formulations (a-c)and PLF-7 formulations (d-e), respectively, by the external addition ofthe respective amounts of Avicel® PH 101 or 102, Explotab®, Mg stearate,Pearlitol® 200 SD, reported in Table 8.

PLF-6 (a-c) and PLF-7 (d-e) were compressed into circular, biconvex 650mg tablets. A ten-station rotary compression machine (Riddhi Pharmamachinery Ltd., Ahmedabad, India) was used for preparing the tablets bydirect compression. Avicel® PH 102 was used as an excipient for directcompression. Mannitol (Pearlitol® SD 200) was used with the intent ofincreasing tablet dispersion and thereby dissolution. The dissolutionprofiles of the tablets were compared with that of capsule formulations.

The in vitro dissolutions of PLF-6 (a-c) and PLF-7 (d) were carried outusing type II apparatus at 50 rpm in 900 ml of PBS pH 6.80. However, thedissolution of PLF-7 (e) was carried out at 75 rpm. Samples werecollected at the time intervals reported in FIG. 13 to analyze therelease of testosterone from the formulations. The analyses wereperformed according to the HPLC assay method described in ComparativeExample 2.

The comparison of formulations that contained either Avicel® PH 101 orAvicel® PH 102 showed similar results with respect to dissolution.Generally, an increase in agitation speed resulted in an faster rate ofdissolution. These studies also showed that formulations containing 20%Pearlitol® (w/w) demonstrated improved dissolution as compared toformulations containing 10% Pearlitol®.

TABLE 8 PLF-6 (a) PLF-6 (b) PLF-6 (c) PLF-7 (d) PLF-7 (e) IngredientsTablet Tablet Tablet Tablet Tablet PLF-6   240 mg 240 mg   240 mg — —3.0 g Testosterone 2.7 g DSPC 0.3 g Cholesterol PLF-7 — — —   360 mg  240 mg 1.2 g Testosterone 1.080 g DSPC 0.12 g Cholesterol Pearlitol ®200 SD — 130 mg   65 mg — — Avicel ® PH 101   388 mg — — —   388 mgAvicel ® PH 102 — 259 mg   323 mg   268 mg — Explotab ® 3 wt % 19.50 mg19.50 mg   19.50 mg 19.50 mg 19.50 mg Mg stearate 1.625 mg 1.625 mg  1.625 mg 1.625 mg 1.625 mg

Example 8 Comparison of Dissolution of Formulations in the Form ofCapsule, Tablet and Powder

The dissolutions of a capsule form of PLF-2 and a tablet form of PLF-6were compared to each other and to the dissolution of unformulatedtestosterone (T1-175). PLF-2 and PLF-6 were prepared as described inExamples 5 and 7, respectively, and further formulated by the externaladdition of excipients as reported in Table 9. Dissolution offormulations in the form of capsule, tablet were compared with that ofpure drug in PBS (pH 6.80) with 1% (w/v) SLS. The addition of 1% (w/v)SLS showed complete drug release within 0.5 h. The in vitro releaseprofiles are shown in FIG. 14. Formulations a, b, and c in Table 9correlated with lines a. b, and c of FIG. 14.

TABLE 9 a b c Ingredients Capsule 650 mg tablet Pure Drug Testosterone —— 50 mg PLF-2 100 mg — — PLF-6 —   240 mg — Avicel ® PH 101 200 mg   388mg — Explotab ® 3 wt % — 19.50 mg — Magnesium stearate — 1.625 mg —

Example 9 Dissolution of Testosterone/Cholesterol DSPC ProliposomalFormulations with Avicel® PH 102 and Explotab® Disintegrant

Capsule formulations PLF-9 (a-e) were prepared by the external additionof Avicel® PH 102 and Explotab® disintegrant, as reported in Table 10,including two formulations were prepared with a minimum amount ofAvicel®, both with and without Explotab® disintegrant. Compositions ofall the formulations described in this example are listed in Table 10.Dissolution of all these formulations was carried out using a type IIapparatus with a capsule sinker in PBS (pH 6.80) medium that did notcontain SLS. The objective of these studies was to determine whether theincrease in ratio of Avicel® to testosterone would increase thesolubility of testosterone from proliposomal formulations.

The in vitro release profiles of the formulations listed in Table 10 areshown in FIG. 15. Formulations with a base formulation: Avicel® ratio of1:1 correlated to greater testosterone release. The other preparationsthat contained minimum amounts of Avicel® showed less testosteronerelease due to poor dispersion of the formulations. The compositions ofPLF-9 and PLF-2 were the same, except the batch size for PLF-9 wasscaled to 5 g, rather than the 3 g batch size of PLF-2.

TABLE 10 PLF-9 (a) PLF-9 (b) PLF-9 (c) PLF-9 (d) PLF-9 (e) Vcaps ® sizeVcaps ® size Vcaps ® size Vcaps ® Plus Vcaps ® Plus Ingredients ‘00’‘00’ ‘00’ size ‘0’ size ‘00’ PLF-9 120 mg 120 mg 120 mg 240 mg 240 mgAvicel ® PH 102 240 mg 60 mg 120 mg  40 mg  40 mg (1:2) (1:0.5) (1:1)(1:0.6) (1:0.6) Explotab ® — — — —  8.5 mg

Example 10 Dissolution of Testosterone/Cholesterol DSPC ProliposomalFormulations with Minimum Amounts of Avicel® PH102, and Explotab®Disintegrant in HPMC Size ‘0’ Capsules

HPMC size ‘0’ capsule formulations PLF-11 (a and b) and PLF-12 (c and d)were prepared by the external addition of Avicel® PH 102 and Explotab®disintegrant, as reported in Table 11 to PLF-10, which was preparedaccording to the same protocol used to make PLF-2 as described inExample 2. All of these formulations contained minimum amounts ofAvicel®, both with and without Explotab® disintegrant. The formulationsin Table 11 were filled into Vcaps® Plus hypromellose (HPMC) capsules(Capsugel, Belgium Nev.) In vitro dissolution studies were carried outusing a type II apparatus at 75 rpm. The dissolution media used was: a)PBS with 1% (w/v) SLS; b) PBS with 2% (w/v) SLS; c) PBS with 0.5% (w/v)SLS; and d) PBS with 1% (w/v) SLS. Media containing 1% and 2% (w/v) SLSshowed complete drug release within 2 hours. The in vitro dissolutionprofiles of formulations (a-d) are shown in FIG. 16.

TABLE 11 PLF-12 © PLF-12 (d) PLF-11 (a) PLF-11 (b) Vcaps ® Vcaps ®Vcaps ® Plus Vcaps ® Plus Plus Plus Ingredients size ‘0’ size ‘0’ size‘0’ size ‘0’ PLF-10 240 mg 240 mg 240 mg 240 mg Avicel ®  40 mg  40 mg 40 mg  40 mg PH 102 Explotab ® — —  8.5 mg  8.5 mg

Example 11 Dissolution Behaviour of Testosterone/Cholesterol DSPCProliposomal Formulations with Avicel® PH 102 in Size ‘0’ Capsules

The formulations PLF-14 (a) and PLF-15 (b), which are provided in Table12 were prepared to determine the disintegration times of formulationsfilled in Vcaps (HPMC) size ‘0’ and Vcaps® Plus (HPMC) size ‘0’ capsules(Capsugel, Belgium Nev.). Formulation PLF-14 was also prepared tocompare disintegration of Vcaps® versus Vcaps® Plus encapsulatedformulations. Size ‘0’ capsules were used to assess each capsule type.To perform the comparison study of Vcaps® and Vcaps Plus®, the capsuleswere filled with only Avicel®.

Formulation PLF-15, which contained the base testosterone, DSPC, andcholesterol components of PLF-13, Avicel®, and Explotab® in the amountsreported in Table 12, was used to compare dispersion times in thepresence and absence of a sinker. A sinker is a basket like device madeof a few turns of platinum wire that is used to prevent capsules fromfloating. These studies revealed that dispersion of capsules was morecomplete when a sinker was not used because the capsules were notconfined within the sinker cage, and because the formulation wereexposed to a larger surface area. See Table 12.

TABLE 12 (b) (a) PLF-15 PLF-14 Vcaps size ‘0’ Vcaps size ‘0’ Vcaps sizeVcaps Plus Sinker used for Dissolved using a Ingredients ‘0’ size ‘0’dissolution sinker PLF-13 — — 240 mg 240 mg Contains Testosterone, DSPC,and Cholesterol in ratios of 1:9:0.1 Avicel ® PH 102 288 mg  40 mg  40mg Explotab ® —  8.5 mg  8.5 mg Disintegration disintegrate disintegrate10% of the capsule 90% of capsule d in 8-11 min d in 11-13 min dispersesin 30 min. gets dispersed in 30 min. DT & dispersion is fast

Example 12 Dissolution of Testosterone/Cholesterol DSPC ProliposomalFormulations with Avicel® PH 102, Prosolv® SMCC 90, DCP, and Explotab®Disintegrant

The base formulation, PLF-13, was further formulated to make PLF-16 (a),PLF-17 (b), PLF-18 (c), PLF-19 (d), PLF-20 (e), PLF-21 (f), and PLF (g)by the external addition of various amounts of different diluentsAvicel® PH 102, Prosolv® SMCC 90 (Silicified Micro CrystallineCellulose), and dibasic calcium phosphate (DCP) in combination withdisintegrants. The formulations were prepared using the ingredientsdescribed in Table 13. PLF-13 contained testosterone, DSPC, andCholesterol in a ratio of 1:0.9:0.1, which correlated with 60 mg, 54 mg,and 6 mg, respectively, per capsule. PLF-13 was prepared as described inExample 2 for PLF-2, except that the preparation of PLF-13 was scaled toa 10 g batch size. The formulations in Table 13 were filled in Vcaps®size ‘0’ HPMC capsules (Capsugel, Belgium Nev.). The in vitrodissolution was carried out using type II apparatus at 75 rpm in 1000 mlof PBS pH 6.80 with 0.5% SLS. No sinker was used in these studies.Formulations containing higher amounts of Avicel® PH 102 and DCP showedbetter release profiles (FIG. 17).

TABLE 13 PLF-16 PLF-17 PLF-18 PLF-19 PLF-20 PLF-21 PLF-22 Ingredients(a) (b) (c) (d) (e) (f) (g) PLF-13  120 mg  120 mg  120 mg  120 mg 120mg  120 mg  120 mg DCP — — —   75 mg — —   30 mg Avicel ® PH 102   75 mg  50 mg 93.40 — Prosolv ®   75 mg   30 mg — SMCC 90 Explotab ® 4.25 mg4.25 mg 4.25 mg 4.25 mg  6.6 mg 4.65 mg 4.65 mg Capsule type Vcaps VcapsVcaps Vcaps Vcaps Vcaps Vcaps & size size ‘0’ size ‘0’ size ‘0’ size ‘0’size ‘0’ size ‘0’ size ‘0’

Example 13 Dissolution of Enteric Coated Capsules ContainingTestosterone/Cholesterol DSPC Proliposomal Formulations with Avicel® PH102, and Explotab® Disintegrant

A base proliposomal formulation was prepared according to thedescription of the preparation of PLF-2 in Example 2, except that thebatch size was scaled to 15 g. The base PLF-2 formulation was furtherformulated into PLF-25 by the external addition of excipients asreported in Table 14, and filled into Vcap® HPMC size ‘00’ capsules(Capsulate, Belgium Nev.). Formulation PLF-24, which is also describedin Table 14, served as a placebo control that did not containtestosterone. PLF-24 and PLF-25 were prepared as part of an effort tooptimize the delayed release coating (e.g., an enteric coating) processfor capsules of the invention.

After the PLF-24 and PLF-25 formulations were filled into capsules, thecapsules were coated with an Eudragit® L 30D-55 polymer-based entericcoating composition. Coating of the capsules was accomplished by using aProCepT® pan coating machine (Zelzate, Belgium) and a Caleva® minicoater air suspension coating machine (Dorset, UK). The Eudragit® L30D-55 coating composition was used in accordance with itsmanufacturer's instructions and the coating process parameters aresummarized in Tables 15 and 16. The capsules of the testosteroneformulation were coated until the capsules experienced a 10.16% weightgain. The placebo formulation was coated until the capsules experiencedan 11.06% weight gain. Based on these results, of the coatingexperiments, the percentage gain in weight for enteric coated capsuleswas fixed to 12%, which provided sufficient resistance to tabletdissolution in acidic pH. For the scale up formulations, Eudragit® L 10055, which is available in powder form, was used for delayed releasecoating (e.g., an enteric coating).

The method used for in vitro dissolution was based on a method describedin the United States Pharmacopeia (USP) 30, <711> Dissolution procedurefor delayed release dosage forms (method B) that was modified by adding0.5% (w/v) SLS for the dissolutions of delayed release dosage forms likeenteric coated capsules. The method involves two stages of testing, theAcid stage and the Buffer stage. In the Acid stage, the dissolution wascarried out in 1000 ml of 0.1N HCl, and maintained at 37±0.5° C. for 2hours. After 2 hours, a sample aliquot was withdrawn to be used in thebuffer stage. In the Buffer stage of testing, phosphate buffer that hasbeen previously equilibrated to 37±0.5° C. was used. The acid wasdrained from the vessel and 1000 ml of pH 6.8 phosphate buffer, preparedby mixing 0.1 N HCl with 0.20 M tribasic sodium phosphate (3:1) andadjusting if necessary with 2 N HCL or 2 N sodium hydroxide, was addedto the vessel. The apparatus ran for 4 hours, and sample aliquots werewithdrawn at regular time intervals. The samples were analyzed using asuitable analytical technique. This modified method was used to testformulations PLF-24 and PLF-25. The dissolution medium used in thebuffer stage was 1000 ml of phosphate buffer solution (PBS) pH 6.80 with0.5% (w/v) SLS. PLF-24 and PLF-25 were intact in acidic pH for 2 hours.The capsules of active formulation showed complete drug release within 2hours in PBS with 0.5% (w/v) SLS (FIG. 18).

A modified and validated HPLC assay method was used for the analysis ofdissolution samples. HPLC analysis was carried out using mobile phasecontaining methanol: water (60:40 v/v). Separation was achieved on aC18; 150×4.6 mm (5 μm) (Ace) column. The mobile phase flow rate was setat 1.2 ml/min while the column temperature was maintained at 25° C. Thetotal run time was 15 minutes with injection volume of 35 μl. The drugwas detected using a UV detector at absorbance maxima of 246 nm. Theretention time of testosterone was found to be 11.5 minutes. The methodwas able to resolve testosterone and all other excipients. The flow ratewas kept high to reduce the run time for each sample to facilitate fastanalysis.

TABLE 14 (a) PLF-24 Placebo (b) PLF-25 Ingredients Enteric coatedcapsule Enteric coated capsule PLF-2 No PLF-2 240 mg 108 mg DSPC 12 mgcholesterol Avicel ® PH 102 152 mg 152 mg Explotab ® 12 mg 12 mg Totalweight 0.285 g 0.405 g Dosage form Vcaps ® size ‘00’ Vcaps ® size ‘00’ %weight gain (w/w, 11.06% 10.16% increase/original total) of entericcoated capsules using Eudragit ® L 30D-55

TABLE 15 Parameter Value Drum speed 12 rpm Atomizing air pressure 1.5bar Pressure drop cabinet 50.0 mBar Inlet air temperature 40° C. Outletair temperature 50° C. Dosing speed 0.6 ml/min Pump speed 35

TABLE 16 Parameter Value Agitation frequency 25 Hz Air inlet temperature40° C. Pump Flow rate 4 rpm Air pressure 1 bar Fan speed 16 m/sec

Example 14 Dissolution of Enteric Coated Capsules ContainingTestosterone/Cholesterol DSPC Proliposomal Formulations with Avicel® PH102, Prosolv® SMCC 90/SMCC HD 90, and Explotab® Disintegrant

Base formulations PLF-23 and PLF-28 were further formulated by theexternal addition of excipients, as described in Table 17, including themanual mixing of PLF-23 with two different grades of microcrystallinecellulose (Prosolv® HD 90 and Prosolv® SMCC 90) to make PLF-26 andPLF-27, respectively. PLF-23 and PLF-28 are compositionally identical toeach other and to PLF-2, which is described in Example 2, and containstestosterone, DSPC, and cholesterol in the ratios of 1:0.9:0.1. Themethod used to prepare PLF-23 and PLF-28 was also the same method thatwas used to prepare PLF-2. PLF-28 was further formulated into PLF-29 bythe external addition of excipients, excluding microcrystallinecellulose, as also described in Table 17.

PLF-26 and PLF-27 were filled into Vcaps® HPMC size ‘00’ capsules(Capsulate, Belgium Nev.), and coated with the delayed release coatingpolymer, Eudragit® L 30D-55, according to the coating proceduredescribed in Example 13.

PLF-29 was also filled into capsules, but unlike PLF-26 and PLF-27, itcontained Avicel® PH 102, and remained uncoated. In order to determinethe effect of curing on dissolution, PLF-29 capsules were cured byheating the capsules at 40 C in a hot air oven for two hours

The method used for in vitro dissolution was based on a method describedin the United States Pharmacopeia (USP) 30, <711> Dissolution procedurefor delayed release dosage forms (method B that was modified by adding0.5% (w/v) SLS for the dissolutions of delayed release dosage forms likeenteric coated capsules. The method involves two stages of testing, theAcid stage and the Buffer stage. In the Acid stage, the dissolution wascarried out in 1000 ml of 0.1N HCl, and maintained at 37±0.5° C. for 2hours. After 2 hours, a sample aliquot was withdrawn to be used in thebuffer stage. In the Buffer stage of testing, phosphate buffer that hasbeen previously equilibrated to 37±0.5° C. was used. The acid wasdrained from the vessel and 1000 ml of pH 6.8 phosphate buffer, preparedby mixing 0.1 N HCl with 0.20 M tribasic sodium phosphate (3:1) andadjusting if necessary with 2 N HCL or 2 N sodium hydroxide, was addedto the vessel. The apparatus ran for 4 hours, and sample aliquots werewithdrawn at regular time intervals. The samples were analyzed using asuitable analytical technique. The dissolution medium used in the bufferstage was 1000 ml of phosphate buffer solution (PBS) pH 6.80 with 0.5%(w/v) SLS. Testosterone release from the collected samples wasdetermined using a UV spectrophotometer at a wavelength of 246 nm. FIG.19 shows the in vitro dissolution profiles of formulations (a-c).

TABLE 17 Ingredients PLF-26 (a) PLF-27 (b) PLF-29 (c) PLF-23 240 mgPLF-23 240 mg PLF-23 240 mg PLF-28 PLF-28 Avicel ® PH 102 — — 152 mgProslv ® SMCC 90 — 155 mg — Proslv ® SMCC 155 mg — — HD 90 Explotab ® 10 mg  10 mg  12 mg Total capsule weight 405 mg 405 mg 405 mg Dosageform Vcaps ® Vcaps ® Vcaps ® size ‘00’ size ‘00’ size ‘00’Enteric-coated Enteric-coated Heat cured

In vitro testosterone dissolution studies were carried out usingformulation PLF-28. The dissolution medium used for these studies wasPBS (pH 6.80) with 0.5% (w/v) SLS, and the dissolution protocol followedis described in this Example, above. Samples were collected after 4 h ofdissolution and used for particle size analysis by using a NICOMP (SantaBarbara, Calif.) model 370 sub micron particle size analyzer. Thesesamples were not quantified for percent drug dissolution. Table 18summarizes the results of the particle size analysis of the formulationsas physical mixes and in matrix form after being subjected todissolution.

TABLE 18 Mean diameter of vesicles in nm based on: Number - Volume -Intensity - weighted weighted weighted Gaussian Gaussian Gaussian SampleDetails Sample No. distribution distribution distribution PLF-28 1 216.12061.2 932.0 2 445.1 1369.1 929.9 3 321.1 2873.5 1296.8 Physical mix 1366.0 1961.4 1041.9 (Testosterone + 2 125.7 1999.9 736.3 DSPC + 3 359.63437.4 1362.2 Cholesterol)

Example 15 Formulation and Evaluation of Testosterone Oral DeliverySystem

A. Preparation and Optimization of Proliposomal TestosteroneFormulations Testosterone and phospholipids (Table 19) were dissolved inethanol and the solvent was evaporated using nitrogen gas. The drypowder was passed through #60 mesh screen to produce homogenous particlesize distribution. Phospholipids employed in the study are from InactiveIngredient Guide (FDA).

TABLE 19 Formulation No. Composition Ratio Amount (mg) 1 Test:DMPC 1:1200:200 2 Test:DMPG 1:1 200:200 3 Test:DSPC 1:1 200:200 4 Test:DSPG 1:1200:200 5 Test:(DMPC:DMPG) 1:1 200:(100:100) 6 Test:(DSPC:DMPG) 1:1200:(100:100) 7 Test:(DMPC:Chol) 1:(9:1) 200:(180:20) 8Test:(DMPC:DMPG:) 1:(7:3) 200:(140:60) 9 Test:(DSPC:Chol) 1:(9:1)200:(180:20) 10 Test:(DSPC:DMPG:Chol) 1:(4:4:2) 200:(80:80:40) 11Test:(DMPC:DMPG) 1:(3:7) 200:(60:140) 12 Test:(DSPC:DMPG) 1:(7:3)200:(140:60) 13 Test:(DSPC:DMPG) 1:(3:7) 200:(60:140) 14Test:(DSPC:Chol) 1:(6:4) 200:(120:80) 15 Test:(DSPC:DSPG) 1:(1:1)200:(100:100)

B. Transport Study

Caco-2 Cell Culture:

Monolayers of Caco-2 cells were prepared on 4 m-pore polycarbonateTranswell® filters by the following method. Caco-2 cells were grown inT-75 flasks at 37° C. in an atmosphere of 5% CO₂ and 95% air usingDulbecco's Modified Eagle Medium (DMEM, pH 7.2) with the necessarygrowth supplements. The medium was changed at least once prior to 90%confluence. The cells were washed with Hank's Balanced Salt Solution(HBSS w/o Ca⁺², Mg⁺²) and trypsinized with 0.25% trypsin in 1 mM EDTAfor 5 min. at 37° C. The cells were resuspended in 10 ml of DMEM andprocessed to minimize aggregation of cells. Five ml of the cellsuspension were withdrawn and seeded into 4-μm pore Transwell® insertsat a density of 7.5×10 cells/ml. Five ml of DMEM was added to the flaskto make a 1:1 dilution of the cell suspension to DMEM, and the cellswere re-seeded. The cells in the Transwell® inserts were grown forapproximately five days and the resistance of the cells was measuredevery other day until resistance was greater than 100Ω.

C. In Vitro Transport Studies

Once the resistance of the cells reached greater than 100Ω the DMEM wascarefully aspirated and replaced with 1.5 ml HBSS w/Ca⁺², Mg⁺² (whichhas been warmed in the 37° C. water bath) in the donor compartment and2.5 ml in the receiver compartment using a micro pipette and allowed toequilibrate at room temperature for 30 minutes. Then the HBSSw/Ca⁺²,Mg⁺² was carefully aspirated from the donor compartment andreplaced with 0.5 mg/ml formulation which had been resuspended in HBSSw/Ca⁺², Mg⁺² (placed in 37° C. water bath for 30 minutes) prepared theprevious day. Each formulation was tested in triplicates. The resistancewas measured for each well (to ensure the cells stay intact) and 300 μlof sample was withdrawn from the receiver compartment and compensatedwith 300 μl of fresh HBSS w/Ca⁺², Mg⁺² for each time. Samples werewithdrawn at times 5, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270,300 minutes. The amount of testosterone transported was determined byanalysis of 100 μl of the sample by HPLC. The experiments were performedin triplicate. Controls: 1. Unformulated testosterone control; 2.Testosterone proliposomal formulations

Transport Study Results:

Transport of testosterone across Caco-2 cells after 5 hrs is shown inFIG. 20. The transport of testosterone was greater with all fifteenproliposomal formulations compared to control (processed andnon-processed testosterone). Formulations prepared with DMPG andDSPC:Chol (9:1) exhibited the maximum transport of testosterone amongthe fifteen formulations evaluated in this study. The transport oftestosterone was 2-fold greater than control (processed andnon-processed testosterone). At the end of 5 hr sampling time, thetransport had not reached plateau. This suggests the possibility offurther enhancement of transport of testosterone beyond 5 hrs.

D. Conclusion

Addition of Cholesterol to DSPC facilitates improved transport oftestosterone across the intestinal membrane. The improved transport oftestosterone across the membrane was surprising positive result.

Example 16 Pharmacokinetics Studies

A clinical study was carried out that involved ten human subjects withhypogonadism. Medical examination was carried out prior to recruitingthe patients for the study. The study was carried out in both fed andfasted conditions using a clinical study protocol that was approved bythe Institutional Animal Care and Use Committee (IACUC). Informedconsent was obtained from all the patients prior to the start of thestudy. Pharmacokinetics of testosterone after oral administration of thedrug in two different doses (120 & 240 mg) was compiled. The mean plasmaconcentration of testosterone and its major metabolite Dihydrotestosterone (DHT) under different treatment conditions is given in FIG.21, 22. Absorption of testosterone was delayed in presence of food.Administration of higher dose of testosterone did not show any linearincrease in the plasma concentration of testosterone. The peak plasmaconcentration (C_(max)) of testosterone was found after 5 hours(C_(max)) of administration of the drug for both strengths.

Example 17 Oral Dosage Form Containing Testoterone/Cholesterol DSPCProliposomal Formulation

An oral dosage form in the form of an enteric coated capsule wasprepared on pilot scale for clinical study. The capsule contained, on aper capsule basis, a pharmaceutical composition having the ingredientslisted in Table 20.

The oral dosage form was prepared by the following method using thecomponents set out in Table 20. The method of preparing the oral dosageform is appropriate for the ranges indicated in Table 20 for eachcomponent provided by the table. The process involved dissolvingtestosterone in alcohol to get a clear solution. DSPC was added to thedrug solution followed by addition of cholesterol to form a dispersion.The solvent was removed by evaporation using a rota evaporator undervacuum. Distillation until a dry mass was obtained. The dried lumps werethen passed through sieve #60 and blended with microcrystallinecellulose and sodium starch glycolate. The formulation was filled intosize ‘00’ capsules using a semi automated capsule filling machine. Thecapsules were enteric coated using a methacrylic acid copolymer C.

TABLE 20 Components/Composition of Oral Dosage Form Core Components(capsule): % wt range Function Testosterone USP 12.5-60.0  Activeingredient Distearoylphosphatidylcholine (DSPC) 13.33-53.2  PhospholipidCholesterol 2.96-11.84 Neutrolipid Microcrystalline Cellulose NF(PH-102) 9.87-37.74 Filler/binder Sodium Starch Glycolate NF 1-5 Disintegrant Alcohol USP* — Solvent Purified Water USP* — Solvent

Example 18 Clinical Study of the Pharmacokinetics of Testosterone inHuman Subjects with Hypogonadism

A clinical study was carried out in 34 human subjects with hypogonadismto evaluate testosterone pharmacokinetics of over the course of their 15day treatment periods with an oral dosage form of testosterone thatcontained the per-capsule amounts of the components provided in Table21, and prepared according to the method described in Example 17.Subjects with ≦300 ng/dL serum testosterone were considered ashypogonadal. The inclusion criteria for all men to participate in thestudy was to have body mass index (BMI)<39 Kg/m², more than 18 years inage and the weight requirement was ≧55 Kg. All the subjects understoodthe purpose of the study and signed the informed consent form prior toparticipating in the study. The subject group was divided into twogroups of 17 that were each administered twice-daily (i.e., morning andevening doses) dosages of either 120 mg or 240 mg (i.e., two 120 mgdoses administered at the same time) of the formulated testosterone for15 days. Plasma samples were taken on an hourly basis from the subjectsafter one full day and at 15 days after the dosing regimen began.

TABLE 21 Core Components (capsule): Wt per capsule Function TestosteroneUSP 120.0 mg Active ingredient Distearoylphosphatidylcholine 108.0 mgPhospholipid (DSPC) Cholesterol  12.0 mg Neutrolipid MicrocrystallineCellulose NF 152.85 mg  Filler/binder (Avicel ® PH-102) Sodium StarchGlycolate NF 12.15 mg Disintegrant (Explotab ®) Alcohol* q.s Total 405.0mg HPMC capsules size ‘00’ 120.0 mg Enteric Coating composition (forcapsule): Methacrylic Acid Copolymer  39.5 mg Enteric coating C. NF,polymer Triethyl Citrate NF  3.95 mg Plasticizer Talc NF 19.55 mg Antitacking agent Purified water* q.s. Diluent Total weight 588.0 mg

FIG. 23 shows the separate averages of the combined hourly plasmatestosterone concentrations from subjects on the 120 mg and 240 mg twicedaily regimens over the course of the first and fifteenth days oftreatment. As FIG. 23 indicates, twice-daily administration of the 240mg dose of testosterone did not show any linear increase in the plasmaconcentration of testosterone than that of the twice-daily 120 mgregimen. On Day 1, the 120 mg and 240 mg dosing regimens each achievedaverage daily plasma testosterone concentrations of greater than 300ng/dL in 71% of the subjects. On Day 15, the 120 mg and 240 mg dosingregimens achieved average daily plasma testosterone concentrations ofgreater than 300 ng/dL in 59% and 31% of the subjects, respectively.From day 1 to day 15 of the study, there were decreases in testosteroneexposure for the subjects that were administered the 120 mg and 240 mgdosing regimens. The decrease associated with the 240 mg regimen wassignificant.

The average peak plasma concentration (C_(max)) of testosterone wasreached four hours after the administration of either the 120 mg or 240mg doses, at which point it was 500 ng/dL and greater than 500 ng/dL,respectively. See FIGS. 24 and 25.

Example 19 Evaluation of the Toxicity and Toxicokinetics of ProliposomalDosage Forms

The purpose of this study was to evaluate the toxicity andtoxicokinetics of an oral dosage form of testosterone that contained theper-capsule amounts of the components provided in Table 21, and preparedaccording to the method described in Example 17. For these studies, aplacebo, and three different doses of formulated testosterone wereadministered once daily to beagle dogs for a minimum of 90 consecutivedays. More specifically, 16 male beagle dogs were assigned to fourtreatment groups in the toxicology portion of this study. Animals weredosed orally with capsules providing a target dose level of 0, 15, 75,or 150 mg/kg/day of formulated testosterone for 91 consecutive days.These dose levels are equivalent to o, 120 mg, 600 mg, and 1200 mgdosage forms. On day 92, animals were euthanized and subjected to acomplete gross necropsy. Protocol-specified tissues, including testes,were collected and forwarded to Experimental Pathology Laboratories(EPL), Inc. Tissues from all groups were processed, embedded inparaffin, sectioned, and stained with hematoxylin and eosin (H&E). Theresulting slides were forwarded to Brett Saladino, DVM, Diplomate ACVPof Calvert Laboratories for evaluation by light microscopy.

The only pathological findings that were attributed to the pharmacologiceffects of exogenous testosterone on the testes and epididymides.Findings included hypospermatogenesis, increased multinucleate germcells/syncytia, increased luminal spermatogenic cells,vacuolation/atrophy of Leydig cells, and tubular atrophy in the testes;and hypospermia and increased luminal debris in the epididymides. Thefindings were most striking in animals given 75 or 150 mg/ml/day.

Blood samples were also drawn at fixed time intervals and analyzed ondays 1, 57 and 91 for 24 hour periods on each of those days. Bloodsamples were collected from all animals prior to treatment initiationand from study animals on days 29 and 92 via jugular vein puncture. SeeFIGS. 26, 27, and 28, respectively.

The examples and embodiments described herein are illustrative andvarious modifications or changes suggested to persons skilled in the artare to be included within this disclosure. As will be appreciated bythose skilled in the art, the specific components listed in the aboveexamples may be replaced with other functionally equivalent components.While an increase in plasma testosterone levels increased with increasesin the amount of testosterone dosage, the increase was not proportional.Testosterone levels increased on day 57 compared to day 1. The levelsincreased with all three doses (120, 600 and 1200 mg). Testosteronelevels decreased on day 91 compared to day 57. In case of 120 mg dosethe testosterone level was lower than day 1.

What is claimed is:
 1. A method of testosterone replacement therapy(TRT) to treat hypogonadism in an individual in need thereof, comprisingadministering a proliposomal powder dispersion consisting essentially of(a) native testosterone, (b) cholesterol, and (c) at least onephospholipid, wherein (a) and (b) are present in a weight ratio (a):(b)ranging from 1.0:0.05 to 1.0:0.30 and (a), (b) and (c) are present in aweight ratio of (a):((b)+(c)) ranging from 1.0:1.0 and 1.0:2.5, whereinthe amount of the proliposomal powder dispersion administered to theindividual is sufficient to raise the individual's serum testosteroneconcentration to at least 300 ng/dL.
 2. The method of TRT according toclaim 1, wherein the phospholipid is selected from distearoylphosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoylphosphatidylcholine, egg phosphatidylcholine, soy phosphatidylcholine,dimyristyl phosphatidyl glycerol sodium, 1,2-dimyristoyl-phosphatidicacid, dipalmitoylphosphatidylglycerol, dipalmitoyl phosphate,1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol,1,2-distearoyl-sn-glycero-3-phosphatidic acid, phosphatidylserine andsphingomyelin, or a combination thereof.
 3. The method TRT according toclaim 2, wherein the phospholipid is distearoyl phosphatidylcholine. 4.The method of TRT according to claim 1, wherein the proliposomal powderdispersion is administered in an oral dosage form.
 5. The method of TRTaccording to claim 4, wherein the oral dosage form comprises thedispersion, admixed with microcrystalline cellulose, and the dispersionand the microcrystalline cellulose are present in a weight ratio rangingfrom about 1:0.5 to about 1:2.
 6. The method of TRT according to claim5, wherein the microcrystalline cellulose are present in a weight ratioranging from 1:0.5 to 1:1.
 7. The method of TRT according to claim 1 orclaim 4, wherein the dosage of the native testosterone is an amount from50 to 260 mg.
 8. The method of TRT according to claim 1 or claim 4,wherein the dosage amount administered to the human in need thereof is120 mg, administered twice daily.
 9. The method of TRT according toclaim 1 or claim 4, wherein the dosage amount administered to the humanin need thereof is 240 mg, twice daily.
 10. The method of TRT accordingto claim 4, wherein the oral dosage form is a capsule coated with adelayed release coating.
 11. The method of TRT according to claim 1 orclaim 4, wherein the individual in need thereof has a serum testosteroneconcentration of 300 ng/dL or less.
 12. The method of TRT according toclaim 1 or claim 4, wherein the hypogonadism is associated withtestosterone deficiency syndrome.
 13. The method of TRT according toclaim 1 or claim 4, wherein the hypogonadism is associated with androgendeficiency of the aging male (ADAM).